Compounds and methods for modulating cell adhesion

ABSTRACT

Cyclic peptides and compositions comprising such cyclic peptides are provided. The cyclic peptides comprise a cadherin cell adhesion recognition sequence HAV. Methods for using such peptides and compositions for modulating cadherin-mediated cell adhesion in a variety of contexts are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/996,679,filed Dec. 23, 1997 now pending, which is a continuation in part of U.S.Ser. No. 08/893,534, filed Jul. 11, 1997 now U.S. Pat. No. 6,031,072,which claims the benefit of U.S. Provisional Application No. 60/021,612,filed on Jul. 12, 1996, now abandoned.

TECHNICAL FIELD

The present invention relates generally to methods for modulating celladhesion, and more particularly to cyclic peptides comprising a cadherincell adhesion recognition sequence, and to the use of such cyclicpeptides for inhibiting or enhancing cadherin-mediated cell adhesion.

BACKGROUND OF THE INVENTION

Cell adhesion is a complex process that is important for maintainingtissue integrity and generating physical and permeability barrierswithin the body. All tissues are divided into discrete compartments,each of which is composed of a specific cell type that adheres tosimilar cell types. Such adhesion triggers the formation ofintercellular junctions (i.e., readily definable contact sites on thesurfaces of adjacent cells that are adhering to one another), also knownas tight junctions, gap junctions and belt desmosomes. The formation ofsuch junctions gives rise to physical and permeability barriers thatrestrict the free passage of cells and other biological substances fromone tissue compartment to another. For example, the blood vessels of alltissues are composed of endothelial cells. In order for components inthe blood to enter a given tissue compartment, they must first pass fromthe lumen of a blood vessel through the barrier formed by theendothelial cells of that vessel. Similarly, in order for substances toenter the body via the gut, the substances must first pass through abarrier formed by the epithelial cells of that tissue. To enter theblood via the skin, both epithelial and endothelial cell layers must becrossed.

Cell adhesion is mediated by specific cell surface adhesion molecules(CAMs). There are many different families of CAMs, including theimmunoglobulin, integrin, selectin and cadherin superfamilies, and eachcell type expresses a unique combination of these molecules. Cadherinsare a rapidly expanding family of calcium-dependent CAMs (Munro et al.,In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp.17-34, RG Landes Co.(Austin Tex., 1996). The classical cadherins(abbreviated CADs) are integral membrane glycoproteins that generallypromote cell adhesion through homophilic interactions (a CAD on thesurface of one cell binds to an identical CAD on the surface of anothercell), although CADs also appear to be capable of forming heterotypiccomplexes with one another under certain circumstances and with loweraffinity. Cadherins have been shown to regulate epithelial, endothelial,neural and cancer cell adhesion, with different CADs expressed ondifferent cell types. N (neural)—cadherin is predominantly expressed byneural cells, endothelial cells and a variety of cancer cell types. E(epithelial)—cadherin is predominantly expressed by epithelial cells.Other CADs are P (placental)—cadherin, which is found in human skin andR (retinal)-cadherin. A detailed discussion of the classical cadherinsis provided in Munro SB et al., 1996, In: Cell Adhesion and Invasion inCancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, AustinTex.).

The structures of the CADs are generally similar. As illustrated in FIG.1, CADs are composed of five extracellular domains (EC1-EC5), a singlehydrophobic domain (TM) that transverses the plasma membrane (PM), andtwo cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN(SEQ ID NO:8), DXD and LDRE (SEQ ID NO:9) are interspersed throughoutthe extracellular domains. The first extracellular domain (EC1) containsthe classical cadherin cell adhesion recognition (CAR) sequence, HAV(His-Ala-Val), along with flanking sequences on either side of the CARsequence that may play a role in conferring specificity. Syntheticpeptides containing the CAR sequence and antibodies directed against theCAR sequence have been shown to inhibit CAD-dependent processes (Munroet al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuket al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell.Physiol. 156:610-18, 1993). The three-dimensional solution and crystalstructures of the EC1 domain have been determined (Overduin et al.,Science 267:386-389, 1995; Shapiro et al., Nature 374:327-337, 1995).

Although cell adhesion is required for certain normal physiologicalfunctions, there are situations in which cell adhesion is undesirable.For example, many pathologies (such as autoimmune and inflammatorydiseases) involve abnormal cellular adhesion. Cell adhesion may alsoplay a role in graft rejection. In such circumstances, modulation ofcell adhesion may be desirable.

In addition, permeability barriers arising from cell adhesion createdifficulties for the delivery of drugs to specific tissues and tumorswithin the body. For example, skin patches are a convenient tool foradministering drugs through the skin. However, the use of skin patcheshas been limited to small, hydrophobic molecules because of theepithelial and endothelial cell barriers. Similarly, endothelial cellsrender the blood capillaries largely impermeable to drugs, and theblood/brain barrier has hampered the targeting of drugs to the centralnervous system. In addition, many solid tumors develop internal barriersthat limit the delivery of anti-tumor drugs and antibodies to innercells.

Attempts to facilitate the passage of drugs across such barriersgenerally rely on specific receptors or carrier proteins that transportmolecules across barriers in vivo. However, such methods are ofteninefficient, due to low endogenous transport rates or to the poorfunctioning of a carrier protein with drugs. While improved efficiencyhas been achieved using a variety of chemical agents that disrupt celladhesion, such agents are typically associated with undesirableside-effects, may require invasive procedures for administration and mayresult in irreversible effects. It has been suggested that linearsynthetic peptides containing a cadherin CAR sequence may be employedfor drug transport (WO 91/04745), but such peptides are oftenmetabolically unstable and are generally considered to be poortherapeutic agents.

Accordingly, there is a need in the art for compounds that modulate celladhesion and improve drug delivery across permeability barriers withoutsuch disadvantages. The present invention fulfills this need and furtherprovides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides cyclic peptides and methods formodulating cadherin-mediated cell adhesion. Within one aspect, thepresent invention provides cyclic peptides comprising the sequenceHis-Ala-Val, wherein the cyclic peptides modulate cadherin-mediated celladhesion. Within one embodiment a cyclic peptide has the formula:

wherein X₁, and X₂ are optional, and if present, are independentlyselected from the group consisting of amino acid residues andcombinations thereof in which the residues are linked by peptide bonds,and wherein X₁ and X₂ independently range in size from 0 to 10 residues,such that the sum of residues contained within X₁ and X₂ ranges from 1to 12; wherein Y₁ and Y₂ are independently selected from the groupconsisting of amino acid residues, and wherein a covalent bond is formedbetween residues Y₁ and Y2; and wherein Z₁ and Z₂ are optional, and ifpresent, are independently selected from the group consisting of aminoacid residues and combinations thereof in which the residues are linkedby peptide bonds. Such cyclic peptides may comprise modifications suchas an N-acetyl or N-alkoxybenzyl group and/or a C-terminal amide orester group. Cyclic peptides may be cyclized via, for example, adisulfide bond; an amide bond between terminal functional groups,between residue side-chains or between one terminal functional group andone residue side chain; a thioether bond or δ₁δ₁-ditryptophan, or aderivative thereof.

Within further aspects, the present invention provides cell adhesionmodulating agents that comprise a cyclic peptide as described above.Within specific embodiments, such modulating agents may be linked to oneor more of a targeting agent, a drug, a solid support or supportmolecule, or a detectable marker. In addition, or alternatively, a celladhesion modulating agent may further comprising one or more of: (a) acell adhesion recognition sequence that is bound by an adhesion moleculeother than a cadherin, wherein the cell adhesion recognition sequence isseparated from any HAV sequence(s) by a linker; and/or (b) an antibodyor antigen-binding fragment thereof that specifically binds to a celladhesion recognition sequence bound by an adhesion molecule other than acadherin.

The present invention further provides pharmaceutical compositionscomprising a cell adhesion modulating agent as described above, incombination with a pharmaceutically acceptable carrier. Suchcompositions may further comprise a drug. Alternatively, or in addition,such compositions may comprise: (a) a peptide comprising a cell adhesionrecognition sequence that is bound by an adhesion molecule other than acadherin; and/or (b) an antibody or antigen-binding fragment thereofthat specifically binds to a cell adhesion recognition sequence bound byan adhesion molecule other than a cadherin.

Within further aspects, methods are provided for modulating celladhesion, comprising contacting a cadherin-expressing cell with a celladhesion modulating agent as described above. Within certainembodiments, methods are provided for modulating N-cadherin mediatedcell adhesion, comprising contacting an N-cadherin-expressing cell witha cell adhesion modulating agent comprising a cyclic peptide having theformula:

wherein Y is optional and, if present is selected from the groupconsisting of amino acid residues and combinations thereof in which theresidues are linked by peptide bonds, and wherein Y ranges in size from0 to 10 residues; and wherein X and Z are independently selected fromthe group consisting of amino acid residues, wherein a disulfide bond isformed between residues X and Z; and wherein X comprises an N-acetylgroup.

Within a further aspect, methods are provided for reducing unwantedcellular adhesion in a mammal, comprising administering to a mammal acell adhesion modulating agent as described above, wherein themodulating agent inhibits cadherin-mediated cell adhesion.

In a further aspect, a method is provided for enhancing the delivery ofa drug to a tumor in a mammal, comprising administering to a mammal acell adhesion modulating agent as described above and a drug, whereinthe modulating agent inhibits cadherin-mediated cell adhesion.

Within related aspects, methods for treating cancer and/or inhibitingmetastasis of tumor cells in a mammal are provided, comprisingadministering to a mammal afflicted with cancer a cell adhesionmodulating agent as described above, wherein the modulating agentinhibits cadherin-mediated cell adhesion.

In a further aspect, methods are provided for inducing apoptosis in acadherin-expressing cell, comprising contacting a cadherin-expressingcell with a cell adhesion modulating agent as described above, whereinthe modulating agent inhibits cadherin-mediated cell adhesion.

The present invention also provides, within other aspects, methods forinhibiting angiogenesis in a mammal, comprising administering to amammal a cell adhesion modulating agent as described above, wherein themodulating agent inhibits cadherin-mediated cell adhesion.

Within a further embodiment, the present invention provides methods forenhancing drug delivery to the central nervous system of a mammal,comprising administering to a mammal a cell adhesion modulating agent asdescribed above, wherein the modulating agent inhibits cadherin-mediatedcell adhesion.

In still further aspects, methods are provided for enhancing celladhesion. Within one such aspect, methods for enhancing wound healing ina mammal are provided, comprising contacting a wound in a mammal with acell adhesion modulating agent as described above, wherein themodulating agent enhances cadherin-mediated cell adhesion.

Within a related aspect, the present invention provides methods forenhancing adhesion of foreign tissue implanted within a mammal,comprising contacting a site of implantation of foreign tissue in amammal with a cell adhesion modulating agent as described above, whereinthe modulating agent enhances cadherin-mediated cell adhesion.

In a further aspect, the present invention provides methods for treatinga demyelinating neurological disease in a mammal, comprisingadministering to a mammal a cell adhesion modulating agent as describedabove, wherein the modulating agent inhibits cadherin-mediated celladhesion.

Within a related aspect, the present invention provides methods forfacilitating migration of an N-cadherin expressing cell on astrocytes,comprising contacting an N-cadherin expressing cell with (a) a celladhesion modulating agent that inhibits cadherin-mediated cell adhesion,wherein the modulating agent comprises a cyclic peptide that comprisesthe sequence HAV; and (b) one or more astrocytes; and therebyfacilitating migration of the N-cadherin expressing cell on theastrocytes.

The present invention also provides methods for modulating the immunesystem of a mammal, comprising administering to a mammal a cell adhesionmodulating agent as described above, wherein the modulating agentinhibits cadherin-mediated cell adhesion.

In yet another aspect, methods for preventing pregnancy in a mammal areprovided, comprising administering to a mammal a cell adhesionmodulating agent as described above, wherein the modulating agentinhibits cadherin-mediated cell adhesion.

Within a further aspect, methods are provided for increasingvasopermeability in a mammal, comprising administering to a mammal acell adhesion modulating agent as described above, wherein themodulating agent inhibits cadherin-mediated cell adhesion.

The present invention further provides methods for inhibiting synapticstability in a mammal, comprising administering to a mammal a celladhesion modulating agent as described above, wherein the modulatingagent inhibits cadherin-mediated cell adhesion.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.All references disclosed herein are hereby incorporated by reference intheir entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the structure of classical CADs. The fiveextracellular domains are designated EC1-EC5, the hydrophobic domainthat transverses the plasma membrane (PM) is represented by TM, and thetwo cytoplasmic domains are represented by CP1 and CP2. The calciumbinding motifs are shown by DXNDN (SEQ ID NO:8), DXD and LDRE (SEQ IDNO:9). The CAR sequence, HAV, is shown within EC1. Cytoplasmic proteinsβ-catenin (β), α-catenin (α) and α-actinin (ACT), which mediate theinteraction between CADs and microfilaments (MF) are also shown.

FIG. 2 provides the amino acid sequences of mammalian classical cadherinEC1 domains: human N-cadherin (SEQ ID NO:1), mouse N-cadherin (SEQ IDNO:2), cow N-cadherin (SEQ ID NO:3), human P-cadherin (SEQ ID NO:4),mouse P-cadherin (SEQ ID NO:5), human E-cadherin (SEQ ID NO:6) and mouseE-cadherin (SEQ ID NO:7).

FIGS. 3A-3I provides the structures of representative cyclic peptides ofthe present invention (structures on the left hand side), along withsimilar, but inactive, structures (on the right).

FIG. 4 is a histogram depicting the mean neurite length in microns forneurons grown in the presence (solid bars) or absence (cross-hatchedbars) of 500 μg/mL of the representative cyclic peptide N-Ac-CHAVC-NH₂(SEQ ID NO: 10). In the first pair of bars, neurons were grown on amonolayer of untransfected 3T3 cells. In the remaining columns, the meanneurite length is shown for neurons cultured on 3T3 cells transfectedwith cDNA encoding N-CAM (second pair of bars), L1 (third pair of bars)or N-cadherin (fourth pair of bars).

FIGS. 5A-5C are photographs showing monolayer cultures of bovineendothelial cells in the presence (FIG. 5A) and absence (FIG. 5C) of arepresentative cyclic peptide or in the presence of an inactive controlpeptide (FIG. 5B). FIG. 5A shows the cells 30 minutes after exposure to500 μg/mL N-Ac-CHAVC-NH₂ (SEQ ID NO:10). FIG. 5B shows the cells 30minutes after exposure to the control peptide N-Ac-CHGVC-NH₂ (SEQ IDNO:11). FIG. 5C shows the cells in the absence of cyclic peptide. Notethat the endothelial cells retracted from one another in the presence ofN-Ac-CHAVC-NH₂ (SEQ ID NO: 10).

FIGS. 6A-6C are photographs showing monolayer cultures of bovineendothelial cells in the presence (FIG. 6A) and absence (FIG. 6C) of arepresentative cyclic peptide or in the presence of an inactive controlpeptide (FIG. 6B). FIG. 6A shows the cells 30 minutes after exposure to500 μg/mL N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24). FIG. 6B shows the cells 30minutes after exposure to the control peptide N-Ac-CAHGVDIC-NH₂ (SEQ IDNO:25). FIG. 6C shows the cells in the absence of cyclic peptide. Inthis case, neither of the cyclic peptides show activity.

FIGS. 7A-7C are photographs showing monolayer cultures of bovineendothelial cells in the presence (FIG. 7A) and absence (FIG. 7C) of arepresentative cyclic peptide or in the presence of an inactive controlpeptide (FIG. 7B). FIG. 7A shows the cells 30 minutes after exposure to500 μg/mL N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26). FIG. 7B shows the cells 30minutes after exposure to the control peptide N-Ac-CAHGVDC-NH₂ (SEQ IDNO:27). FIG. 7C shows the cells in the absence of cyclic peptide. Notethat the endothelial cells retracted from one another in the presence ofN-Ac-CAHAVDC-NH₂ (SEQ ID NO:26).

FIGS. 8A-8C are photographs showing monolayer cultures of bovineendothelial cells in the presence (FIG. 8A) and absence (FIG. 8C) of arepresentative cyclic peptide or in the presence of an inactive controlpeptide (FIG. 8B). FIG. 8A shows the cells 30 minutes after exposure to500 pg/mL N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42). FIG. 8B shows the cells 30minutes after exposure to the control peptide N-Ac-CSHGVSSC-NH₂ (SEQ IDNO:43). FIG. 8C shows the cells in the absence of cyclic peptide. Notethat the endothelial cells retracted from one another and round up inthe presence of N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42).

FIGS. 9A-9F are photographs showing monolayer cultures of human ovariancancer cells (SKOV3) in the presence (FIGS. 9A and D-F) and absence(FIG. 9C) of a representative cyclic peptide or in the presence of aninactive control peptide (FIG. 9B). FIG. 9A shows the cells 24 hoursafter being cultured in the presence of 500 μg/mL N-Ac-CHAVC-NH₂ (SEQ IDNO:10; 10× magnification). FIG. 9B shows the cells (10× magnification)24 hours after being cultured in the presence of the control peptideN-Ac-CHGVC-NH₂ (SEQ ID NO:11). FIG. 9C shows the cells (10×magnification) in the absence of cyclic peptide. FIGS. 9D-F show thecells (20× magnification) 48 hours after exposure to N-Ac-CHAVC-NH₂ (SEQID NO:10) at concentrations of 1 mg/mL, 100 μg/mL and 10 μg/mL,respectively. Note that the SKOV3 cells retract from one another andround-up when cultured in the presence of either 0.5 or 1 mg/mlN-Ac-CHAVC-NH₂ (SEQ ID NO: 10).

FIGS. 10A and 10B are photographs showing monolayer cultures of humanovarian cancer cells (SKOV3) 24 hours after exposure to 500 μg/mL of therepresentative cyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO: 10) (FIG. 10A)or the control peptide N-Ac-CHGVC-NH₂ (SEQ ID NO:11) (FIG. 10B). Notethat the SKOV3 cells round-up when cultured in the presence of 0.5 mg/mlN-Ac-CHAVC-NH₂ (SEQ ID NO: 10).

FIGS. 11A-11D are photographs of monolayer cultures of normal rat kidney(NRK) cells untreated (FIG. 11A) or after 48 hours of exposure to 1mg/mL H-CHAVSC-OH (SEQ ID NO:38) (FIG. 11B), the control peptideN-Ac-CHGVC-NH₂ (SEQ ID NO:11), (FIG. 11C) or the representative cyclicpeptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10), (FIG. 11D). Note that NRK cellsretract from one another when cultured in the presence of N-Ac-CHAVC-NH₂(SEQ ID NO:10). Furthermore the NRK cells do not form cobblestone-likemonolayers when exposed to this peptide.

FIGS. 12A-12D are immunofluorescence photographs of the monolayer normalrat kidney (NRK) cultures shown in FIGS. 11A-D immunolabeled forE-cadherin. FIG. 12A shows untreated cells and FIGS. 12B-D show cellsafter 48 hours of exposure to either 1 mg/mL H-CHAVSC-OH (SEQ ID NO:38)(FIG. 12B), the control peptide N-Ac-CHGVC-NH₂ (SEQ ID NO:11), (FIG.12C) or the representative cyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10),(FIG. 12D). Note that E-cadherin expression is greatly reduced in thecells treated with N-Ac-CHAVC-NH₂ (SEQ ID NO:10), as compared to theE-cadherin levels expressed by untreated cells and cells treated withthe other two cyclic peptides

FIGS. 13A-13C are photographs showing monolayer cultures of humanovarian cancer cells (OVCAR3) in the presence of varying concentrationsof a representative cyclic peptide. FIG. 13A shows the cells 24 hoursafter being cultured in the presence of 1 mg/ml of N-Ac-CHAVSC-NH₂ (SEQID NO:38). FIG. 13B shows the cells 24 hours after being cultured in thepresence of 100 μg/ml of N-Ac-CHAVSC-NH₂ (SEQ ID NO:38). FIG. 13C showsthe cells 24 hours after being cultured in the presence of 10 μg/ml ofN-Ac-CHAVSC-NH₂ (SEQ ID NO:38). Note that the cells retract form oneanother in the presence of 100 μg/ml of N-Ac-CHAVSC-NH₂ (SEQ ID NO:38),whereas they round up in the presence of 1 mg/ml of this peptide.

FIGS. 14A and 14B are photographs showing cultures of human melanomaME115 cells in the presence (FIG. 14B) and absence (FIG. 14A) of arepresentative cyclic peptide. The cells have been immunolabeled forcadherin. FIG. 14B shows the cells 48 hours after being cultured in thepresence of 500 μg/ml of N-Ac-CHAVC-NH₂ (SEQ ID NO:10). FIG. 14A showsuntreated cultures of human melanoma ME115 cells. Note that cadherin islocalized in intracellular vesicles in cells treated with peptide,whereas it is present at the surface in the untreated cells.

FIGS. 15A and 15B are photographs showing monolayer cultures of A1N4human breast epithelial cells in the presence (FIG. 15B) and absence(FIG. 15A) of a representative cyclic peptide. The cells have beenimmunolabeled for E-cadherin. FIG. 15B shows the cells 48 hours afterbeing cultured in the presence of 500 μg/ml of N-Ac-CHAVC-NH₂ (SEQ IDNO:10). FIG. 15A shows untreated monolayer cultures of A1N4 human breastepithelial cells. Note that the distribution of E-cadherin isnon-contiguous in cells treated with the cyclic peptide. Furthermore,gaps have appeared in the monolayer of cells treated with the peptide.

FIG. 16 is a histogram illustrating the effect of 500 μg/ml of arepresentative cyclic peptide (N-Ac-CHAVC-NH₂; SEQ ID NO:10; treatmentbars) on the penetration of Oregon Green through the skin, as comparedto the effect of the control peptide N-Ac-CHGVC-NH₂ (SEQ ID NO:1;control bars). Penetration was determined by converting fluorescentunits to a concentration unit of microgram/5 ml (volume of the receivercompartment) using a standard curve and regression analysis equations.

FIG. 17 is a histogram illustrating the effect of 500 μg/ml of arepresentative cyclic peptide (N-Ac-CHAVC-NH₂; SEQ ID NO:10; treatmentbars) on the penetration of Rhodamine Green through the skin, ascompared to the effect of the control peptide N-Ac-CHGVC-NH₂ (SEQ IDNO:11; control bars). Penetration was determined by convertingfluorescent units to a concentration unit of microgram/5 ml (volume ofthe receiver compartment) using a standard curve and regression analysisequations.

FIG. 18 is a histogram illustrating the effect of 2.5 mg/ml of arepresentative cyclic peptide (N-Ac-CHAVC-NH₂; SEQ ID NO:10; treatmentbars) on the penetration of Oregon Green through the skin, as comparedto the effect of the control peptide N-Ac-CHGVC-NH₂ (SEQ ID NO:11;control bars). Penetration was determined by converting fluorescentunits to a concentration unit of microgram/5 ml (volume of the receivercompartment) using a standard curve and regression analysis equations.

FIG. 19 is a histogram illustrating the effect of 2.5 mg/ml of arepresentative cyclic peptide (N-Ac-CHAVC-NH₂; SEQ ID NO:10; treatmentbars) on the penetration of Rhodamine Green through the skin, ascompared to the effect of the control peptide N-Ac-CHGVC-NH₂ (SEQ IDNO:11; control bars). Penetration was determined by convertingfluorescent units to a concentration unit of microgram/5 ml (volume ofthe receiver compartment) using a standard curve and regression analysisequations.

FIG. 20 is a graph illustrating the results of a study to assess thechronic toxicity of a representative cyclic peptide. The graph presentsthe mean body weight during the three-day treatment period (oneintraperitoneal injection per day) and the four subsequent recoverydays. Three different doses are illustrated, as indicated.

FIG. 21 is a graph illustrating the stability of a representative cyclicpeptide in mouse whole blood. The percent of the cyclic peptideremaining in the blood was assayed at various time points, as indicated.

FIG. 22 is a bar graph showing the effect of N-Ac-CHAVC-NH₂ (SEQ IDNO:10) and N-Ac-CHGVC-NH₂ (SEQ ID NO:11) on N-cadherin-mediated neuriteoutgrowth. Mean neurite length is shown for cerebellar neurons culturedfor 14 hours on monolayers of control 3T3 cells (unshaded), onN-cadherin expressing 3T3 cells (diagonal rising right), on N-cadherinexpressing 3T3 cells in media supplemented with N-Ac-CHAVC-NH₂ (SEQ IDNO: 10; diagonal cross hatch) and on N-cadherin expressing 3T3 cells inmedia supplemented with N-Ac-CHGVC-NH₂ (SEQ ID NO:11; diagonal risingleft). The results show the mean length of the longest neurite measuredin a single representative experiment, and the error bars show thes.e.m.

FIG. 23 is a graph showing dose-response curves that illustrate theinhibition of neurite outgrowth over both 3T3 cells and N-cadherinexpressing 3T3 cells in the presence of increasing concentrations ofN-Ac-CHAVC-NH₂ (SEQ ID NO:10). The peptide had no effect on the basalgrowth over 3T3 cells. The EC₅₀ value was determined to be 0.22 mM.

FIG. 24 is a bar graph illustrating the effects of the cyclic peptidesN-Ac-CHAVDC-NH₂ (SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50) andN-Ac-CHAVDINC-NH₂ (SEQ ID NO:51) on L1 function. Cerebellar neurons werecultured on monolayers of control 3T3 cells and L1 expressing 3T3 cellsfor 16-18 hours in control media (unshaded) or control mediasupplemented with peptides N-Ac-CHAVDC-NH₂ (SEQ ID NO:20; diagonalrising right), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50; diagonal cross hatch) orN-Ac-CHAVDINC-NH₂ (SEQ ID NO:51; diagonal rising left) at aconcentration of 100 μg/mL. The cultures were then fixed and neuriteoutgrowth determined by measuring the length of the longest neurite froma total of 150-200 neurons sampled in replicate cultures for eachexperimental condition. The results show L1 response, measured as apercentage increase in the mean length of the longest neurite relativeto the 3T3 control value, for neurons grown in the absence or presenceof the test peptide. The results are pooled from three independentexperiments, and the bars show the s.e.m.

FIG. 25 is a graph dose-response curve that illustrates the inhibitionof neurite outgrowth over N-cadherin expressing 3T3 cells in thepresence of increasing concentrations of N-Ac-CHAVDC-NH₂ (SEQ ID NO:20).

FIG. 26 is a graph dose-response curve that illustrates the inhibitionof neurite outgrowth over N-cadherin expressing 3T3 cells in thepresence of increasing concentrations of N-Ac-CHAVDIC-NH₂ (SEQ IDNO:50).

FIG. 27 is a graph dose-response curve that illustrates the inhibitionof neurite outgrowth over N-cadherin expressing 3T3 cells in thepresence of increasing concentrations of N-Ac-CHAVDINC-NH₂ (SEQ IDNO:51).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides cell adhesion modulatingagents comprising cyclic peptides that are capable of modulatingcadherin-mediated processes, such as cell adhesion. In general, tomodulate cadherin-mediated cell adhesion, a cadherin-expressing cell iscontacted with a cell adhesion modulating agent (also referred to hereinas a “modulating agent”) either in vivo or in vitro. A modulating agentcomprises a cyclic peptide that contains the classical cadherin celladhesion recognition (CAR) sequence HAV (i.e., His-Ala-Val). Suchmodulating agents may further comprise one or more additional CARsequences and/or an antibody (or antigen-binding fragment thereof) thatspecifically binds to a cadherin CAR sequence, as described below.Certain cell adhesion modulating agents (also referred to herein as“modulating agents”) described herein inhibit cell adhesion. Suchmodulating agents may generally be used, for example, to treat diseasesor other conditions characterized by undesirable cell adhesion or tofacilitate drug delivery to a specific tissue or tumor. Alternatively,certain modulating agents may be used to enhance cell adhesion (e.g., tosupplement or replace stitches or to facilitate wound healing) or toenhance or direct neurite outgrowth.

CELL ADHESION MODULATING AGENTS

The term “cell adhesion modulating agent,” as used herein, refers to amolecule comprising at least one cyclic peptide that contains a cadherincell adhesion recognition (CAR) sequence, generally HAV (His-Ala-Val).The term “cyclic peptide,” as used herein, refers to a peptide or saltthereof that comprises (1) an intramolecular covalent bond between twonon-adjacent residues and (2) at least one cadherin CAR sequence. Theintramolecular bond may be a backbone to backbone, side-chain tobackbone or side-chain to side-chain bond (ie., terminal functionalgroups of a linear peptide and/or side chain functional groups of aterminal or interior residue may be linked to achieve cyclization).Preferred intramolecular bonds include, but are not limited to,disulfide, amide and thioether bonds. In addition to the cadherin CARsequence HAV, a modulating agent may comprise additional CAR sequences,which may or may not be cadherin CAR sequences, and/or antibodies orfragments thereof that specifically recognize a CAR sequence. AdditionalCAR sequences may be present within the cyclic peptide containing theHAV sequence, within a separate cyclic peptide component of themodulating agent and/or in a non-cyclic portion of the modulating agent.Antibodies and antigen-binding fragments thereof are typically presentin a non-cyclic portion of the modulating agent.

Within certain embodiments, a cyclic peptide preferably comprises anN-acetyl group (i.e., the amino group present on the amino terminalresidue of the peptide prior to cyclization is acetylated). It has beenfound, within the context of the present invention, that the presence ofsuch an acetyl group may enhance cyclic peptide activity for certainapplications.

In addition to the CAR sequence(s), cyclic peptides generally compriseat least one additional residue, such that the size of the cyclicpeptide ring ranges from 4 to about 15 residues, preferably from 5 to 10residues. Such additional residue(s) may be present on the N-terminaland/or C-terminal side of a CAR sequence, and may be derived fromsequences that flank the HAV sequence within one or more naturallyoccurring cadherins (e.g., N-cadherin, E-cadherin, P-cadherin,R-cadherin or other cadherins containing the HAV sequence) with orwithout amino acid substitutions and/or other modifications. Flankingsequences for endogenous N-, E-, P- and R-cadherin are shown in FIG. 2,and in SEQ ID NOs: 1 to 7. Database accession numbers for representativenaturally occurring cadherins are as follows: human N-cadherin M34064,mouse N-cadherin M31131 and M22556, cow N-cadherin X53615, humanP-cadherin X63629, mouse P-cadherin X06340, human E-cadherin Z13009,mouse E-cadherin X06115. Alternatively, additional residues present onone or both sides of the CAR sequence(s) may be unrelated to anendogenous sequence (e.g., residues that facilitate cyclization).

Within certain preferred embodiments, as discussed below, relativelysmall cyclic peptides that do not contain significant sequences flankingthe HAV sequence are preferred for modulating N-cadherin and E-cadherinmediated cell adhesion. Such peptides may contain an N-acetyl group anda C-amide group (e.g., the 5-residue ring N-Ac-CHAVC-NH₂ (SEQ ID NO:10)or N-Ac-KHAVD-NH₂ (SEQ ID NO:12)). The finding, within the presentinvention, that such relatively small cyclic peptides may be effectiveand all-purpose inhibitors of cell adhesion represents a unexpecteddiscovery. Such cyclic peptides can be thought of as “master keys” thatfit into peptide binding sites of each of the different classicalcadherins, and are capable of disrupting cell adhesion of neural cells,endothelial cells, epithelial cells and/or certain cancer cells. Smallcyclic peptides may generally be used to specifically modulate celladhesion of neural and/or other cell types by topical administration orby systemic administration, with or without linking a targeting agent tothe peptide, as discussed below.

Within other preferred embodiments, a cyclic peptide may containsequences that flank the HAV sequence on one or both sides that aredesigned to confer specificity for cell adhesion mediated by one or morespecific cadherins, resulting in tissue and/or cell-type specificity.Suitable flanking sequences for conferring specificity include, but arenot limited to, endogenous sequences present in one or more naturallyoccurring cadherins, and cyclic peptides having specificity may beidentified using the representative screens provided herein. Forexample, it has been found, within the context of the present invention,that cyclic peptides that contain additional residues derived from thenative E-cadherin sequence on the C-terminal side of the CAR sequenceare specific for epithelial cells (i.e., such peptides disruptE-cadherin mediated cell adhesion to a greater extent than they disruptN-cadherin expression). The addition of appropriate endogenous sequencesmay similarly result in peptides that disrupt N-cadherin mediated celladhesion. For example, it has been found within the context of thepresent invention that the addition of one or more amino acid residueson the C-terminal side of the HAV sequence in an endogenous N-cadherinresults in cyclic peptides that are potent inhibitors of neuriteoutgrowth.

To facilitate the preparation of cyclic peptides having a desiredspecificity, nuclear magnetic resonance (NMR) and computationaltechniques may be used to determine the conformation of a peptide thatconfers a known specificity. NMR is widely used for structural analysisof molecules. Cross-peak intensities in nuclear Overhauser enhancement(NOE) spectra, coupling constants and chemical shifts depend on theconformation of a compound. NOE data provide the interproton distancebetween protons through space and across the ring of the cyclic peptide.This information may be used to facilitate calculation of the lowestenergy conformation for the HAV sequence. Conformation may then becorrelated with tissue specificity to permit the identification ofpeptides that are similarly tissue specific or have enhanced tissuespecificity.

As noted above, multiple CAR sequences may be present within amodulating agent. CAR sequences that may be included within a modulatingagent are any sequences specifically bound by an adhesion molecule. Asused herein, an “adhesion molecule” is any molecule that mediates celladhesion via a receptor on the cell's surface. Adhesion moleculesinclude members of the cadherin gene superfamily that are not classicalcadherins (e.g., proteins that do not contain an HAV sequence and/or oneor more of the other characteristics recited above for classicalcadherins), such as desmogleins (Dsg) and desmocollins (Dsc); integrins;members of the immunoglobulin supergene family, such as N-CAM; and otheruncategorized transmembrane proteins, such as occludin, as well asextracellular matrix proteins such as laminin, fibronectin, collagens,vitronectin, entactin and tenascin. Preferred CAR sequences forinclusion within a modulating agent include (a) Arg-Gly-Asp (RGD), whichis bound by integrins (see Cardarelli et al., J. Biol. Chem.267:23159-64, 1992); (b) Tyr-Ile-Gly-Ser-Arg (YIGSR; SEQ ID NO:52),which is bound by α6β1 integrin; (c) KYSFNYDGSE (SEQ ID NO:53), which isbound by N-CAM; (d) the N-CAM heparin sulfate-binding siteIWKHKGRDVILKKDVRF (SEQ ID NO:54); (e) the occludin CAR sequence LYHY(SEQ ID NO:55); (f) claudin CAR sequences comprising at least fourconsecutive amino acids present within a claudin region that has theformula: Trp-Lys/Arg-Aaa-Baa-Ser/Ala-Tyr/Phe-Caa-Gly (SEQ ID NO:56),wherein Aaa, Baa and Caa indicate independently selected amino acidresidues; Lys/Arg is an amino acid that is lysine or arginine; Ser/Alais an amino acid that is serine or alanine; and Tyr/Phe is an amino acidthat is tyrosine or phenylalanine; and (g) nonclassical cadherin CARsequences comprising at least three consecutive amino acids presentwithin a nonclassical cadherin region that has the formula:Aaa-Phe-Baa-Ile/Leu/Val-Asp/Asn/Glu-Caa-Daa-Ser/Thr/Asn-Gly (SEQ IDNO:57), wherein Aaa, Baa, Caa and Daa are independently selected aminoacid residues; Ile/Leu/Val is an amino acid that is selected from thegroup consisting of isoleucine, leucine and valine, Asp/Asn/Glu is anamino acid that is selected from the group consisting of aspartate,asparagine and glutamate; and Ser/Thr/Asn is an amino acid that isselected from the group consisting of serine, threonine or asparagine.Representative claudin CAR sequences include IYSY (SEQ ID NO:58), TSSY(SEQ ID NO:59), VTAF (SEQ ID NO:60) and VSAF (SEQ ID NO:61).Representative nonclassical cadherin CAR sequences include theVE-cadherin (cadherin-5) CAR sequence DAE; the cadherin-6 CAR sequencesEEY, NEN, ESE and DSG; the cadherin-7 CAR sequences DEN, EPK and DAN;the cadherin-8 CAR sequences EEF and NDV; the OB-cadherin (cadherin-11)CAR sequences DDK, EEY and EAQ; the cadherin-12 CAR sequences DET andDPK; the cadherin-14 CAR sequences DDT, DPK and DAN; the cadherin-15 CARsequences DKF and DEL; the PB-cadherin CAR sequences EEY, DEL, DPK andDAD; the protocadherin CAR sequences DLV, NRD, DPK and DPS; the dsg CARsequences NQK, NRN and NKD; the dsc CAR sequences EKD and ERD and thecadherin-related neuronal receptor CAR sequences DPV, DAD, DSV, DSN,DSS, DEK and NEK.

Linkers may, but need not, be used to separate CAR sequences and/orantibody sequences within a modulating agent. Linkers may also, oralternatively, be used to attach one or more modulating agents to asupport molecule or material, as described below. A linker may be anymolecule (including peptide and/or non-peptide sequences as well assingle amino acids or other molecules), that does not contain a CARsequence and that can be covalently linked to at least two peptidesequences. Using a linker, HAV-containing cyclic peptides and otherpeptide or protein sequences may be joined head-to-tail (i.e., thelinker may be covalently attached to the carboxyl or amino group of eachpeptide sequence), head-to-side chain and/or tail-to-side chain.Modulating agents comprising one or more linkers may form linear orbranched structures. Within one embodiment, modulating agents having abranched structure comprise three different CAR sequences, such as RGD,YIGSR (SEQ ID NO:52) and HAV, one or more of which are present within acyclic peptide. Within another embodiment, modulating agents having abranched structure comprise RGD, YIGSR (SEQ ID NO:52), HAV andKYSFNYDGSE (SEQ ID NO:53). In a third embodiment, modulating agentshaving a branched structure comprise HAV and LYHY (SEQ ID NO:55), alongwith one or more of NQK, NRN, NKD, EKD and ERD. Bi-functional modulatingagents that comprise an HAV sequence with flanking E-cadherin-specificsequences joined via a linker to an HAV sequence with flankingN-cadherin-specific sequences are also preferred for certainembodiments.

Linkers preferably produce a distance between CAR sequences between 0.1to 10,000 nm, more preferably about 0.1-400 nm. A separation distancebetween recognition sites may generally be determined according to thedesired function of the modulating agent. For inhibitors of celladhesion, the linker distance should be small (0.1-400 nm). Forenhancers of cell adhesion, the linker distance should be 400-10,000 nm.One linker that can be used for such purposes is (H₂N(CH₂)_(n)CO₂H)_(m),or derivatives thereof, where n ranges from 1 to 10 and m ranges from 1to 4000. For example, if glycine (H₂NCH₂CO₂H) or a multimer thereof isused as a linker, each glycine unit corresponds to a linking distance of2.45 angstroms, or 0.245 nm, as determined by calculation of its lowestenergy conformation when linked to other amino acids using molecularmodeling techniques. Similarly, aminopropanoic acid corresponds to alinking distance of 3.73 angstroms, aminobutanoic acid to 4.96angstroms, aminopentanoic acid to 6.30 angstroms and amino hexanoic acidto 6.12 angstroms. Other linkers that may be used will be apparent tothose of ordinary skill in the art and include, for example, linkersbased on repeat units of 2,3-diaminopropanoic acid, lysine and/orornithine. 2,3-Diaminopropanoic acid can provide a linking distance ofeither 2.51 or 3.11 angstroms depending on whether the side-chain aminoor terminal amino is used in the linkage. Similarly, lysine can providelinking distances of either 2.44 or 6.95 angstroms and ornithine 2.44 or5.61 angstroms. Peptide and non-peptide linkers may generally beincorporated into a modulating agent using any appropriate method knownin the art.

Modulating agents that inhibit cell adhesion typically contain one HAVsequence or multiple HAV sequences, which may be adjacent to one another(i.e., without intervening sequences) or in close proximity (i.e.,separated by peptide and/or non-peptide linkers to give a distancebetween the CAR sequences that ranges from about 0.1 to 400 nm). Withinone such embodiment, the cyclic peptide contains two HAV sequences. Sucha modulating agent may additionally comprise a CAR sequence for one ormore different adhesion molecules (including, but not limited to, otherCAMs) and/or one or more antibodies or fragments thereof that bind tosuch sequences. Linkers may, but need not, be used to separate such CARsequence(s) and/or antibody sequence(s) from the HAV sequence(s) and/oreach other. Such modulating agents may generally be used within methodsin which it is desirable to simultaneously disrupt cell adhesionmediated by multiple adhesion molecules. Within certain preferredembodiments, the second CAR sequence is derived from fibronectin and isrecognized by an integrin (i.e., RGD; see Cardarelli et al., J. Biol.Chem. 267:23159-23164, 1992)., or is an occludin CAR sequence (e.g.,LYHY; SEQ ID NO:55). One or more antibodies, or fragments thereof, maysimilarly be used within such embodiments.

Modulating agents that enhance cell adhesion may contain multiple HAVsequences, and/or antibodies that specifically bind to such sequences,joined by linkers as described above. Enhancement of cell adhesion mayalso be achieved by attachment of multiple modulating agents to asupport molecule or material, as discussed further below. Suchmodulating agents may additionally comprise one or more CAR sequence forone or more different adhesion molecules (including, but not limited to,other CAMs) and/or one or more antibodies or fragments thereof that bindto such sequences, to enhance cell adhesion mediated by multipleadhesion molecules.

Modulating agents and cyclic peptides as described herein may compriseresidues of L-amino acids, D-amino acids, or any combination thereof.Amino acids may be from natural or non-natural sources, provided that atleast one amino group and at least one carboxyl group are present in themolecule; α- and β-amino acids are generally preferred. The 20 L-aminoacids commonly found in proteins are identified herein by theconventional three-letter or one-letter abbreviations indicated in Table1, and the corresponding D-amino acids are designated by a lower caseone letter symbol. Modulating agents and cyclic peptides may alsocontain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Preferred derivatives include amino acids having an N-acetylgroup (such that the amino group that represents the N-terminus of thelinear peptide prior to cyclization is acetylated) and/or a C-terminalamide group (i.e., the carboxy terminus of the linear peptide prior tocyclization is amidated). Residues other than common amino acids thatmay be present with a cyclic peptide include, but are not limited to,penicillamine, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine,β-mercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, α-aminoadipic acid, m-aminomethylbenzoic acid andα,β-diaminopropionic acid.

TABLE 1 Amino acid one-letter and three-letter abbreviations A AlaAlanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C CysCysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H HisHistidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met Methionine FPhe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W TrpTryptophan Y Tyr Tyrosine V Val Valine

Modulating agents and cyclic peptides as described herein may besynthesized by methods well known in the art, including recombinant DNAmethods and chemical synthesis. Chemical synthesis may generally beperformed using standard solution phase or solid phase peptide synthesistechniques, in which a peptide linkage occurs through the directcondensation of the α-amino group of one amino acid with the α-carboxygroup of the other amino acid with the elimination of a water molecule.Peptide bond synthesis by direct condensation, as formulated above,requires suppression of the reactive character of the amino group of thefirst and of the carboxyl group of the second amino acid. The maskingsubstituents must permit their ready removal, without inducing breakdownof the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (see Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy.The Boc strategy uses a 1% cross-linked polystyrene resin. The standardprotecting group for α-amino functions is the tert-butyloxycarbonyl(Boc) group. This group can be removed with dilute solutions of strongacids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid istypically coupled to the amino acyl resin using dicyclohexylcarbodiimide(DCC). Following completion of the assembly, the peptide-resin istreated with anhydrous HF to cleave the benzyl ester link and liberatethe free peptide. Side-chain functional groups are usually blockedduring synthesis by benzyl-derived blocking groups, which are alsocleaved by HF. The free peptide is then extracted from the resin with asuitable solvent, purified and characterized. Newly synthesized peptidescan be purified, for example, by gel filtration, HPLC, partitionchromatography and/or ion-exchange chromatography, and may becharacterized by, for example, mass spectrometry or amino acid sequenceanalysis. In the Boc strategy, C-terminal amidated peptides can beobtained using benzhydrylamine or methylbenzhydrylamine resins, whichyield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chainblocking groups and of the peptide-resin link depends upon thedifferences in the rate of acidolytic cleavage. Orthoganol systems havebeen introduced in which the side-chain blocking groups and thepeptide-resin link are completely stable to the reagent used to removethe α-protecting group at each step of the synthesis. The most common ofthese methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach.Within this method, the side-chain protecting groups and thepeptide-resin link are completely stable to the secondary amines usedfor cleaving the N-α-Fmoc group. The side-chain protection and thepeptide-resin link are cleaved by mild acidolysis. The repeated contactwith base makes the Merrifield resin unsuitable for Fmoc chemistry, andp-alkoxybenzyl esters linked to the resin are generally used.Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved by any of a variety oftechniques well known in the art. Within one embodiment, a bond may begenerated between reactive amino acid side chains. For example, adisulfide bridge may be formed from a linear peptide comprising twothiol-containing residues by oxidizing the peptide using any of avariety of methods. Within one such method, air oxidation of thiols cangenerate disulfide linkages over a period of several days using eitherbasic or neutral aqueous media. The peptide is used in high dilution tominimize aggregation and intermolecular side reactions. This methodsuffers from the disadvantage of being slow but has the advantage ofonly producing H₂O as a side product. Alternatively, strong oxidizingagents such as I₂ and K₃Fe(CN)₆ can be used to form disulfide linkages.Those of ordinary skill in the art will recognize that care must betaken not to oxidize the sensitive side chains of Met, Tyr, Trp or His.Cyclic peptides produced by this method require purification usingstandard techniques, but this oxidation is applicable at acid pHs. Byway of example, strong oxidizing agents can be used to perform thecyclization shown below (SEQ ID NOs: 62 and 63), in which the underlinedportion is cyclized:

FmocCysAsp(t-Bu)GlyTyr(t-Bu)ProLys(Boc)Asp(t-Bu)CysLys(t-Bu)Gly-OMe→

FmocCysAsp(t-Bu)GlyTyr(t-Bu)ProLys(Boc)Asp(t-Bu)CysLys(t-Bu)Gly-OMe

Oxidizing agents also allow concurrent deprotection/oxidation ofsuitable S-protected linear precursors to avoid premature, nonspecificoxidation of free cysteine, as shown below (SEQ ID NOs: 64 and 65),where X and Y=S-Trt or S-Acm:

BocCys(X)GlyAsnLeuSer(t-Bu)Thr(t-Bu)Cys(Y)MetLeuGlyOH→

BocCysGlyAsnLeuSer(t-Bu)Thr(t-Bu)CysMetLeuGlyOH

DMSO, unlike I₂ and K₃Fe(CN)₆, is a mild oxidizing agent which does notcause oxidative side reactions of the nucleophilic amino acids mentionedabove. DMSO is miscible with H₂O at all concentrations, and oxidationscan be performed at acidic to neutral pHs with harmless byproducts.Methyltrichlorosilane-diphenylsulfoxide may alternatively be used as anoxidizing agent, for concurrent deprotection/oxidation of S-Acm, S-Tacmor S-t-Bu of cysteine without affecting other nucleophilic amino acids.There are no polymeric products resulting from intermolecular disulfidebond formation. In the example below (SEQ ID NOs: 66 and 67), X is Acm,Tacm or t-Bu:

H-Cys(X)TyrIleGlnAsnCys(X)ProLeuGly-NH₂→

H-CysTyrIleGlnAsnCysProLeuGly-NH₂

Suitable thiol-containing residues for use in such oxidation methodsinclude, but are not limited to, cysteine, β,β-dimethyl cysteine(penicillamine or Pen), β,β-tetramethylene cysteine (Tmc),β,β-pentamethylene cysteine (Pmc), β-mercaptopropionic acid (Mpr),β,β-pentamethylene-β-mercaptopropionic acid (Pmp), 2-mercaptobenzene,2-mercaptoaniline and 2-mercaptoproline. Peptides containing suchresidues are illustrated by the following representative formulas, inwhich the underlined portion is cyclized, N-acetyl groups are indicatedby N-Ac and C-terminal amide groups are represented by —NH₂:

i) N-Ac-Cys-His-Ala-Val-Cys-NH₂ (SEQ ID NO:10)

ii) N-Ac-Cys-Ala-His-Ala-Val-Asp-Ile-Cys-NH₂ (SEQ ID NO:24)

iii) N-Ac-Cys-Ser-His-Ala-Val-Cys-NH₂ (SEQ ID NO:36)

iv) N-Ac-Cys-His-Ala-Val-Ser-Cys-NH₂ (SEQ ID NO:38)

v) N-Ac-Cys-Ala-His-Ala-Val-Asp-Cys-NH₂ (SEQ ID NO:26)

vi) N-Ac-Cys-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:42)

vii) N-Ac-Cys-His-Ala-Val-Ser-Cys-OH (SEQ ID NO:38)

viii) H-Cys-Ala-His-Ala-Val-Asp-Cys-NH₂ (SEQ ID NO:26)

ix) N-Ac-Cys-His-Ala-Val-Pen-NH₂ (SEQ ID NO:68)

x) N-Ac-Ile-Tmc-Tyr-Ser-His-Ala-Val-Ser-Cys-Glu-NH₂ (SEQ ID NO:69)

xi) N-Ac-Ile-Pmc-Tyr-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:70)

xii) Mpr-Tyr-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:71)

xiii) Pmp-Tyr-Ser-His-Ala-Val-Ser-Ser-Cys-NH₂ (SEQ ID NO:72)

xii)

xiii)

It will be readily apparent to those of ordinary skill in the art that,within each of these representative formulas, any of the abovethiol-containing residues may be employed in place of one or both of thethiol-containing residues recited.

Within another embodiment, cyclization may be achieved by amide bondformation. For example, a peptide bond may be formed between terminalfunctional groups (i.e., the amino and carboxy termini of a linearpeptide prior to cyclization). Two such cyclic peptides are AHAVDI (SEQID NO:34) and SHAVSS (SEQ ID NO:46), with or without an N-terminalacetyl group and/or a C-terminal amide. Within another such embodiment,the linear peptide comprises a D-amino acid (e.g., HAVsS; SEQ ID NO:73).Alternatively, cyclization may be accomplished by linking one terminusand a residue side chain or using two side chains, as in KHAVD (SEQ IDNO:12) or KSHAVSSD (SEQ ID NO:48), with or without an N-terminal acetylgroup and/or a C-terminal amide. Residues capable of forming a lactambond include lysine, ornithine (Orn), α-amino adipic acid,m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate oraspartate.

Methods for forming amide bonds are well known in the art and are basedon well established principles of chemical reactivity. Within one suchmethod, carbodiimide-mediated lactam formation can be accomplished byreaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resultingin the formation of an O-acylurea that can be reacted immediately withthe free amino group to complete the cyclization. The formation of theinactive N-acylurea, resulting from O→N migration, can be circumventedby converting the O-acylurea to an active ester by reaction with anN-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide,1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate.In addition to minimizing O→N migration, these additives also serve ascatalysts during cyclization and assist in lowering racemization.Alternatively, cyclization can be performed using the azide method, inwhich a reactive azide intermediate is generated from an alkyl ester viaa hydrazide. Hydrazinolysis of the terminal ester necessitates the useof a t-butyl group for the protection of side chain carboxyl functionsin the acylating component. This limitation can be overcome by usingdiphenylphosphoryl acid (DPPA), which furnishes an azide directly uponreaction with a carboxyl group. The slow reactivity of azides and theformation of isocyanates by their disproportionation restrict theusefulness of this method. The mixed anhydride method of lactamformation is widely used because of the facile removal of reactionby-products. The anhydride is formed upon reaction of the carboxylateanion with an alkyl chloroformate or pivaloyl chloride. The attack ofthe amino component is then guided to the carbonyl carbon of theacylating component by the electron donating effect of the alkoxy groupor by the steric bulk of the pivaloyl chloride t-butyl group, whichobstructs attack on the wrong carbonyl group. Mixed anhydrides withphosphoric acid derivatives have also been successfully used.Alternatively, cyclization can be accomplished using activated esters.The presence of electron withdrawing substituents on the alkoxy carbonof esters increases their susceptibility to aminolysis. The highreactivity of esters of p-nitrophenol, N-hydroxy compounds andpolyhalogenated phenols has made these “active esters” useful in thesynthesis of amide bonds. The last few years have witnessed thedevelopment of benzotriazolyloxytris-(dimethylamino)phosphoniumhexafluorophosphonate (BOP) and its congeners as advantageous couplingreagents. Their performance is generally superior to that of the wellestablished carbodiimide amide bond formation reactions.

Within a further embodiment, a thioether linkage may be formed betweenthe side chain of a thiol-containing residue and an appropriatelyderivatized α-amino acid. By way of example, a lysine side chain can becoupled to bromoacetic acid through the carbodiimide coupling method(DCC, EDAC) and then reacted with the side chain of any of the thiolcontaining residues mentioned above to form a thioether linkage. Inorder to form dithioethers, any two thiol containing side-chains can bereacted with dibromoethane and diisopropylamine in DMF. Examples ofthiol containing linkages are shown below:

Cyclization may also be achieved using δ₁,δ_(1′)-Ditryptophan (i.e.,Ac-Trp-Gly-Gly-Trp-OMe) (SEQ ID NO:74), as shown below:

Representative structures of cyclic peptides are provided in FIG. 3.Within FIG. 3, certain cyclic peptides having the ability to modulatecell adhesion (shown on the left) are paired with similar inactivestructures (on the right). The structures and formulas recited hereinare provided solely for the purpose of illustration, and are notintended to limit the scope of the cyclic peptides described herein.

As noted above, a modulating agent may consist entirely of one or morecyclic peptides, or may contain additional peptide and/or non-peptidesequences, which may be linked to the cyclic peptide(s) usingconventional techniques. Peptide portions may be synthesized asdescribed above or may be prepared using recombinant methods. Withinsuch methods, all or part of a modulating agent can be synthesized inliving cells, using any of a variety of expression vectors known tothose of ordinary skill in the art to be appropriate for the particularhost cell. Suitable host cells may include bacteria, yeast cells,mammalian cells, insect cells, plant cells, algae and other animal cells(e.g., hybridoma, CHO, myeloma). The DNA sequences expressed in thismanner may encode portions of an endogenous cadherin or other adhesionmolecule. Such sequences may be prepared based on known cDNA or genomicsequences (see Blaschuk et al., J. Mol. Biol. 211:679-682, 1990), orfrom sequences isolated by screening an appropriate library with probesdesigned based on the sequences of known cadherins. Such screens maygenerally be performed as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 1989 (and references cited therein). Polymerasechain reaction (PCR) may also be employed, using oligonucleotide primersin methods well known in the art, to isolate nucleic acid moleculesencoding all or a portion of an endogenous adhesion molecule. Togenerate a nucleic acid molecule encoding a peptide portion of amodulating agent, an endogenous sequence may be modified using wellknown techniques. For example, portions encoding one or more CARsequences may be joined, with or without separation by nucleic acidregions encoding linkers, as discussed above. Alternatively, portions ofthe desired nucleic acid sequences may be synthesized using well knowntechniques, and then ligated together to form a sequence encoding aportion of the modulating agent.

As noted above, portions of a modulating agent may comprise an antibody,or antigen-binding fragment thereof, that specifically binds to a CARsequence. As used herein, an antibody, or antigen-binding fragmentthereof, is said to “specifically bind” to a CAR sequence (with orwithout flanking amino acids) if it reacts at a detectable level(within, for example, an ELISA, as described by Newton et al., Develop.Dynamics 197:1-13, 1993) with a peptide containing that sequence, anddoes not react detectably with peptides containing a different CARsequence or a sequence in which the order of amino acid residues in thecadherin CAR sequence and/or flanking sequence is altered.

Antibodies and fragments thereof may be prepared using standardtechniques. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogencomprising a CAR sequence is initially injected into any of a widevariety of mammals (e.g., mice, rats, rabbits, sheep or goats). Smallimmunogens (i.e., less than about 20 amino acids) should be joined to acarrier protein, such as bovine serum albumin or keyhole limpethemocyanin. Following one or more injections, the animals are bledperiodically. Polyclonal antibodies specific for the CAR sequence maythen be purified from such antisera by, for example, affinitychromatography using the modulating agent or antigenic portion thereofcoupled to a suitable solid support.

Monoclonal antibodies specific for a CAR sequence may be prepared, forexample, using the technique of Kohler and Milstein, Eur. J. Immunol.6:511-519, 1976, and improvements thereto. Briefly, these methodsinvolve the preparation of immortal cell lines capable of producingantibodies having the desired specificity from spleen cells obtainedfrom an animal immunized as described above. The spleen cells areimmortalized by, for example, fusion with a myeloma cell fusion partner,preferably one that is syngeneic with the immunized animal. Singlecolonies are selected and their culture supernatants tested for bindingactivity against the modulating agent or antigenic portion thereof.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation, and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target cadherin islocalized.

Within certain embodiments, monoclonal antibodies may be specific forparticular cadherins (e.g., the antibodies bind to E-cadherin, but donot bind significantly to N-cadherin, or vise versa). Such antibodiesmay be prepared as described above, using an immunogen that comprises(in addition to the HAV sequence) sufficient flanking sequence togenerate the desired specificity (e.g., 5 amino acids on each side isgenerally sufficient). One representative immunogen is the 15-merFHLRAHAVDINGNQV-NH₂ (SEQ ID NO:75), linked to KLH (see Newton et al.,Dev. Dynamics 197:1-13, 1993). To evaluate the specificity of aparticular antibody, representative assays as described herein and/orconventional antigen-binding assays may be employed. Such antibodies maygenerally be used for therapeutic, diagnostic and assay purposes, asdescribed herein. For example, such antibodies may be linked to a drugand administered to a mammal to target the drug to a particularcadherin-expressing cell, such as a leukemic cell in the blood.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; see especially page 309) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns (Harlowand Lane, 1988, pages 628-29).

EVALUATION OF MODULATING AGENT ACTIVITY

As noted above, cyclic peptides and other modulating agents as describedherein are capable of modulating (i.e., enhancing or inhibiting)cadherin-mediated cell adhesion. The ability of a modulating agent tomodulate cell adhesion may generally be evaluated in vitro by assayingthe effect of the cyclic peptide on one or more of the following: (1)neurite outgrowth, (2) adhesion between endothelial cells, (3) adhesionbetween epithelial cells (e.g., normal rat kidney cells and/or humanskin) and/or (4) adhesion between cancer cells. In general, a modulatingagent is an inhibitor of cell adhesion if, within one or more of theserepresentative assays, contact of the test cells with the modulatingagent results in a discernible disruption of cell adhesion. Modulatingagents that enhance cell adhesion (e.g., agents comprising multiple HAVsequences and/or linked to a support material) are considered to bemodulators of cell adhesion if they are capable of enhancing neuriteoutgrowth as described below and/or are capable of promoting celladhesion, as judged by plating assays to assess epithelial cell adhesionto a modulating agent attached to a support material, such as tissueculture plastic. For modulating agents that affect N-cadherin mediatedfunctions, assays involving endothelial or cancer cell adhesion orneurite outgrowth are preferred.

Within a representative neurite outgrowth assay, neurons may be culturedon a monolayer of cells (e.g., 3T3) that express N-cadherin. Neuronsgrown on such cells (under suitable conditions and for a sufficientperiod of time) extend longer neurites than neurons cultured on cellsthat do not express N-cadherin. For example, neurons may be cultured onmonolayers of 3T3 cells transfected with cDNA encoding N-cadherinessentially as described by Doherty and Walsh, Curr. Op. Neurobiol.4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994; Hall et al.,Cell Adhesion and Commun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell.Neurosci. 8:99-111, 1994; and Safell et al., Neuron 18:231-242, 1997.Briefly, monolayers of control 3T3 fibroblasts and 3T3 fibroblasts thatexpress N-cadherin may be established by overnight culture of 80,000cells in individual wells of an 8-chamber well tissue culture slide.3000 cerebellar neurons isolated from post-natal day 3 mouse brains maybe cultured for 18 hours on the various monolayers in control media(SATO/2% FCS), or media supplemented with various concentrations of themodulating agent or control peptide. The cultures may then be fixed andstained for GAP43 which specifically binds to the neurons and theirneurites. The length of the longest neurite on each GAP43 positiveneuron may be measured by computer assisted morphometry.

A modulating agent that modulates N-cadherin-mediated cell adhesion mayinhibit or enhance such neurite outgrowth. Under the conditionsdescribed above, the presence of 500 μg/mL of a modulating agent thatdisrupts neural cell adhesion should result in a decrease in the meanneurite length by at least 50%, relative to the length in the absence ofmodulating agent or in the presence of a negative control peptide.Alternatively, the presence of 500 μg/mL of a modulating agent thatenhances neural cell adhesion should result in an increase in the meanneurite length by at least 50%.

Within one representative cell adhesion assay, the addition of amodulating agent to cells that express a cadherin results in disruptionof cell adhesion. A “cadherin-expressing cell,” as used herein, may beany type of cell that expresses at least one cadherin on the cellsurface at a detectable level, using standard techniques such asimmunocytochemical protocols (Blaschuk and Farookhi, Dev. Biol.136:564-567, 1989). Cadherin-expressing cells include endothelial (e.g.,bovine pulmonary artery endothelial cells), epithelial and/or cancercells (e.g., the human ovarian cancer cell line SKOV3 (ATCC #HTB-77)).For example, such cells may be plated under standard conditions thatpermit cell adhesion in the presence and absence of modulating agent(e.g., 500 μg/mL). Disruption of cell adhesion may be determinedvisually within 24 hours, by observing retraction of the cells from oneanother.

For use within one such assay, bovine pulmonary artery endothelial cellsmay be harvested by sterile ablation and digestion in 0.1% collagenase(type II; Worthington Enzymes, Freehold, N.J.). Cells may be maintainedin Dulbecco's minimum essential medium supplemented with 10% fetal calfserum and 1% antibiotic-antimycotic at 37° C. in 7% CO₂ in air. Culturesmay be passaged weekly in trypsin-EDTA and seeded onto tissue cultureplastic at 20,000 cells/cm². Endothelial cultures may be used at 1 weekin culture, which is approximately 3 days after culture confluency isestablished. The cells may be seeded onto coverslips and treated (e.g.,for 30 minutes) with modulating agent or a control compound at, forexample, 500 μg/ml and then fixed with 1% parafornaldehyde. As notedabove, disruption of cell adhesion may be determined visually within 24hours, by observing retraction of the cells from one another. This assayevaluates the effect of a modulating agent on N-cadherin mediated celladhesion.

Within another such assay, the effect of a modulating agent on normalrat kidney (NRK) cells may be evaluated. According to a representativeprocedure, NRK cells (ATCC #1571-CRL) may be plated at 10-20,000 cellsper 35 mm tissue culture flasks containing DMEM with 10% FCS andsub-cultured periodically (Laird et al., J. Cell Biol. 131:1193-1203,1995). Cells may be harvested and replated in 35 mm tissue cultureflasks containing 1 mm coverslips and incubated until 50-65% confluent(24-36 hours). At this time, coverslips may be transferred to a 24-wellplate, washed once with fresh DMEM and exposed to modulating agent at aconcentration of, for example, 1 mg/mL for 24 hours. Fresh modulatingagent may then be added, and the cells left for an additional 24 hours.Cells may be fixed with 100% methanol for 10 minutes and then washedthree times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBSand incubated for a further 1 hour in the presence of mouseanti-E-cadherin antibody (Transduction Labs, 1:250 dilution). Primaryand secondary antibodies may be diluted in 2% BSA/PBS. Followingincubation in the primary antibody, coverslips may be washed three timesfor 5 minutes each in PBS and incubated for 1 hour with donkeyanti-mouse antibody conjugated to fluorescein (diluted 1:200). Followingfurther washes in PBS (3×5 min) coverslips can be mounted and viewed byconfocal microscopy.

In the absence of modulating agent, NRK cells form characteristictightly adherent monolayers with a cobblestone morphology in which cellsdisplay a polygonal shape. NRK cells that are treated with a modulatingagent that disrupts E-cadherin mediated cell adhesion may assume anon-polygonal and elongated morphology (i.e., a fibroblast-like shape)within 48 hours of treatment with 1 mg/mL of modulating agent. Gapsappear in confluent cultures of such cells. In addition, 1 mg/mL of sucha modulating agent reproducibly induces a readily apparent reduction incell surface staining of E-cadherin, as judged by immunofluorescencemicroscopy (Laird et al., J. Cell Biol. 131:1193-1203, 1995), of atleast 75% within 48 hours.

A third cell adhesion assay involves evaluating the effect of a cyclicpeptide on permeability of adherent epithelial and/or endothelial celllayers. For example, the effect of permeability on human skin may beevaluated. Such skin may be derived from a natural source or may besynthetic. Human abdominal skin for use in such assays may generally beobtained from humans at autopsy within 24 hours of death. Briefly, acyclic peptide and a test marker (e.g., the fluorescent markers OregonGreen™ and Rhodamine Green™ Dextran) may be dissolved in a sterilebuffer, and the ability of the marker to penetrate through the skin andinto a receptor fluid may be measured using a Franz Cell apparatus(Franz, Curr. Prob. Dermatol. 7:58-68, 1978; Franz, J. Invest. Dermatol.64:190-195, 1975). In general, a modulating agent that enhances thepermeability of human skin results in a statistically significantincrease in the amount of marker in the receptor compartment after 6-48hours in the presence of 500 μg/mL modulating agent. This assayevaluates the effect of a modulating agent on E-cadherin mediated celladhesion.

MODULATING AGENT MODIFICATION AND FORMULATIONS

A modulating agent as described herein may, but need not, be linked toone or more additional molecules. In particular, as discussed below, itmay be beneficial for certain applications to link multiple modulatingagents (which may, but need not, be identical) to a support molecule(e.g., keyhole limpet hemocyanin) or a solid support, such as apolymeric matrix (which may be formulated as a membrane ormicrostructure, such as an ultra thin film), a container surface (e.g.,the surface of a tissue culture plate or the interior surface of abioreactor), or a bead or other particle, which may be prepared from avariety of materials including glass, plastic or ceramics. For certainapplications, biodegradable support materials are preferred, such ascellulose and derivatives thereof, collagen, spider silk or any of avariety of polyesters (e.g., those derived from hydroxy acids and/orlactones) or sutures (see U.S. Pat. No. 5,245,012). Within certainembodiments, modulating agents and molecules comprising other CARsequence(s) (e.g., an RGD and/or LYHY (SEQ ID NO:55) sequence) may beattached to a support such as a polymeric matrix, preferably in analternating pattern.

Suitable methods for linking a modulating agent to a support materialwill depend upon the composition of the support and the intended use,and will be readily apparent to those of ordinary skill in the art.Attachment may generally be achieved through noncovalent association,such as adsorption or affinity or, preferably, via covalent attachment(which may be a direct linkage between a modulating agent and functionalgroups on the support, or may be a linkage by way of a cross-linkingagent or linker). Attachment of a modulating agent by adsorption may beachieved by contact, in a suitable buffer, with a solid support for asuitable amount of time. The contact time varies with temperature, butis generally between about 5 seconds and 1 day, and typically betweenabout 10 seconds and 1 hour.

Covalent attachment of a modulating agent to a molecule or solid supportmay generally be achieved by first reacting the support material with abifunctional reagent that will also react with a functional group, suchas a hydroxyl, thiol, carboxyl, ketone or amino group, on the modulatingagent. For example, a modulating agent may be bound to an appropriatepolymeric support or coating using benzoquinone, by condensation of analdehyde group on the support with an amine and an active hydrogen onthe modulating agent or by condensation of an amino group on the supportwith a carboxylic acid on the modulating agent. A preferred method ofgenerating a linkage is via amino groups using glutaraldehyde. Amodulating agent may be linked to cellulose via ester linkages.Similarly, amide linkages may be suitable for linkage to other moleculessuch as keyhole limpet hemocyanin or other support materials. Multiplemodulating agents and/or molecules comprising other CAR sequences may beattached, for example, by random coupling, in which equimolar amounts ofsuch molecules are mixed with a matrix support and allowed to couple atrandom.

Although modulating agents as described herein may preferentially bindto specific tissues or cells, and thus may be sufficient to target adesired site in vivo, it may be beneficial for certain applications toinclude an additional targeting agent. Accordingly, a targeting agentmay also, or alternatively, be linked to a modulating agent tofacilitate targeting to one or more specific tissues. As used herein, a“targeting agent,” may be any substance (such as a compound or cell)that, when linked to a modulating agent enhances the transport of themodulating agent to a target tissue, thereby increasing the localconcentration of the modulating agent. Targeting agents includeantibodies or fragments thereof, receptors, ligands and other moleculesthat bind to cells of, or in the vicinity of, the target tissue. Knowntargeting agents include serum hormones, antibodies against cell surfaceantigens, lectins, adhesion molecules, tumor cell surface bindingligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes andthose drugs and proteins that bind to a desired target site. Among themany monoclonal antibodies that may serve as targeting agents areanti-TAC, or other interleukin-2 receptor antibodies; 9.2.27 andNR-ML-05, reactive with the 250 kilodalton human melanoma-associatedproteoglycan; and NR-LU-10, reactive with a pancarcinoma glycoprotein.An antibody targeting agent may be an intact (whole) molecule, afragment thereof, or a functional equivalent thereof. Examples ofantibody fragments are F(ab′)2, -Fab′, Fab and F[v] fragments, which maybe produced by conventional methods or by genetic or proteinengineering. Linkage is generally covalent and may be achieved by, forexample, direct condensation or other reactions, or by way of bi- ormulti-functional linkers. Within other embodiments, it may also bepossible to target a polynucleotide encoding a modulating agent to atarget tissue, thereby increasing the local concentration of modulatingagent. Such targeting may be achieved using well known techniques,including retroviral and adenoviral infection.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds. Theuse of certain specific drugs within the context of the presentinvention is discussed below.

Within certain aspects of the present invention, one or more modulatingagents as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulating agents in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. A modulating agent (alone or incombination with a targeting agent and/or drug) may, but need not, beencapsulated within liposomes using well known technology. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration. For certain topical applications,formulation as a cream or lotion, using well known components, ispreferred.

For certain embodiments, as discussed below, a pharmaceuticalcomposition may further comprise a modulator of cell adhesion that ismediated by one or more molecules other than cadherins. Such modulatorsmay generally be prepared as described above, incorporating one or morenon-cadherin CAR sequences and/or antibodies thereto in place of thecadherin CAR sequences and antibodies. Such compositions areparticularly useful for situations in which it is desirable to inhibitcell adhesion mediated by multiple cell-adhesion molecules, such asother members of the cadherin gene superfamily that are not classicalcadherins (e.g., Dsg and Dsc); claudins; integrins; members of theimmunoglobulin supergene family, such as N-CAM; and other uncategorizedtransmembrane proteins, such as occludin, as well as extracellularmatrix proteins such as laminin, fibronectin, collagens, vitronectin,entactin and tenascin. Preferred CAR sequences for use are as describedabove.

A pharmaceutical composition may also contain one or more drugs, whichmay be linked to a modulating agent or may be free within thecomposition. Virtually any drug may be administered in combination witha cyclic peptide as described herein, for a variety of purposes asdescribed below. Examples of types of drugs that may be administeredwith a cyclic peptide include analgesics, anesthetics, antianginals,antifungals, antibiotics, anticancer drugs (e.g., taxol or mitomycin C),antiinflammatories (e.g., ibuprofen and indomethacin), anthelmintics,antidepressants, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrotubule agents (e.g.,colchicine or vinca alkaloids), antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, anti-signaling agents (e.g.,protein kinase C inhibitors or inhibitors of intracellular calciummobilization), antiarthritics, antithrombin agents, antituberculotics,antitussives, antivirals, appetite suppressants, cardioactive drugs,chemical dependency drugs, cathartics, chemotherapeutic agents,coronary, cerebral or peripheral vasodilators, contraceptive agents,depressants, diuretics, expectorants, growth factors, hormonal agents,hypnotics, immunosuppression agents, narcotic antagonists,parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins(e.g., cholera toxin), tranquilizers and urinary antiinfectives.

For imaging purposes, any of a variety of diagnostic agents may beincorporated into a pharmaceutical composition, either linked to amodulating agent or free within the composition. Diagnostic agentsinclude any substance administered to illuminate a physiologicalfunction within a patient, while leaving other physiological functionsgenerally unaffected. Diagnostic agents include metals, radioactiveisotopes and radioopaque agents (e.g., gallium, technetium, indium,strontium, iodine, barium, bromine and phosphorus-containing compounds),radiolucent agents, contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a colorimetric or fluorometricreaction. In general, such agents may be attached using a variety oftechniques as described above, and may be present in any orientation.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of cyclic peptide followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a cyclic peptidedispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane (see, e.g., European PatentApplication 710,491 A). Carriers for use within such formulations arebiocompatible, and may also be biodegradable; preferably the formulationprovides a relatively constant level of cyclic peptide release. Theamount of cyclic peptide contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented).Appropriate dosages and the duration and frequency of administrationwill be determined by such factors as the condition of the patient, thetype and severity of the patient's disease and the method ofadministration. In general, an appropriate dosage and treatment regimenprovides the modulating agent(s) in an amount sufficient to providetherapeutic and/or prophylactic benefit. Within particularly preferredembodiments of the invention, a modulating agent or pharmaceuticalcomposition as described herein may be administered at a dosage rangingfrom 0.001 to 50 mg/kg body weight, preferably from 0.1 to 20 mg/kg, ona regimen of single or multiple daily doses. For topical administration,a cream typically comprises an amount of modulating agent ranging from0.00001% to 1%, preferably 0.0001% to 0.2%, and more preferably from0.0001% to 0.002%. Fluid compositions typically contain about 10 ng/mlto 5 mg/ml, preferably from about 10 μg to 2 mg/mL cyclic peptide.Appropriate dosages may generally be determined using experimentalmodels and/or clinical trials. In general, the use of the minimum dosagethat is sufficient to provide effective therapy is preferred. Patientsmay generally be monitored for therapeutic effectiveness using assayssuitable for the condition being treated or prevented, which will befamiliar to those of ordinary skill in the art.

MODULATING AGENT METHODS OF USE

In general, the modulating agents and compositions described herein maybe used for modulating the adhesion of cadherin-expressing cells (i.e.,cells that express one or more of E-cadherin, N-cadherin, P-cadherin,R-cadherin and/or other cadherin(s) containing the HAV sequence,including as yet undiscovered cadherins) in vitro and/or in vivo. Asnoted above, modulating agents for purposes that involve the disruptionof cadherin-mediated cell adhesion may comprise a cyclic peptidecontaining a single HAV sequence, multiple HAV sequences in closeproximity and/or an antibody (or an antigen-binding fragment thereof)that recognizes a cadherin CAR sequence. When it is desirable to alsodisrupt cell adhesion mediated by other adhesion molecules, a modulatingagent may additionally comprise one or more CAR sequences bound by suchadhesion molecules (and/or antibodies or fragments thereof that bindsuch sequences), preferably separated from each other and from the HAVsequence by linkers. As noted above, such linkers may or may notcomprise one or more amino acids. For enhancing cell adhesion, amodulating agent may contain multiple HAV sequences or antibodies (orfragments), preferably separated by linkers, and/or may be linked to asingle molecule or to a support material as described above.

Certain methods involving the disruption of cell adhesion as describedherein have an advantage over prior techniques in that they permit thepassage of molecules that are large and/or charged across barriers ofcadherin-expressing cells. As discussed in greater detail below,modulating agents as described herein may also be used to disrupt orenhance cell adhesion in a variety of other contexts. Within the methodsdescribed herein, one or more modulating agents may generally beadministered alone, or within a pharmaceutical composition. In eachspecific method described herein, as noted above, a targeting agent maybe employed to increase the local concentration of modulating agent atthe target site.

In one such aspect, the present invention provides methods for reducingunwanted cellular adhesion by administering a cyclic peptide asdescribed herein. Unwanted cellular adhesion can occur between tumorcells, between tumor cells and normal cells or between normal cells as aresult of surgery, injury, chemotherapy, disease, inflammation or othercondition jeopardizing cell viability or function. Preferred modulatingagents for use within such methods comprise a cyclic peptide such asN-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20),N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ ID NO:22),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30),N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ ID NO:36),N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) andderivatives thereof, including derivatives without the N-acetyl group.In addition, a modulating agent may comprise the sequence RGD, which isbound by integrins, and/or the sequence LYHY (SEQ ID NO:55), which isbound by occludin, separated from the HAV sequence via a linker. OtherCAR sequences that may be present include OB-cadherin, dsg and dsc CARsequences as described above. Alternatively, a separate modulator ofintegrin, occludin-, OB-cadherin-, dsc- and/or dsg-mediated celladhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. Topical administration of the modulating agent(s) isgenerally preferred, but other means may also be employed. Preferably, afluid composition for topical administration (comprising, for example,physiological saline) comprises an amount of cyclic peptide as describedabove, and more preferably an amount ranging from 10 μg/mL to 1 mg/mL.Creams may generally be formulated as described above. Topicaladministration in the surgical field may be given once at the end ofsurgery by irrigation of the wound, as an intermittent or continuousirrigation with use of surgical drains in the post operative period, orby the use of drains specifically inserted in an area of inflammation,injury or disease in cases where surgery does not need to be performed.Alternatively, parenteral or transcutaneous administration may be usedto achieve similar results.

In another aspect, methods are provided for enhancing the delivery of adrug through the skin of a mammal. Transdermal delivery of drugs is aconvenient and non-invasive method that can be used to maintainrelatively constant blood levels of a drug. In general, to facilitatedrug delivery via the skin, it is necessary to perturb adhesion betweenthe epithelial cells (keratinocytes) and the endothelial cells of themicrovasculature. Using currently available techniques, only small,uncharged molecules may be delivered across skin in vivo. The methodsdescribed herein are not subject to the same degree of limitation.Accordingly, a wide variety of drugs may be transported across theepithelial and endothelial cell layers of skin, for systemic or topicaladministration. Such drugs may be delivered to melanomas or may enterthe blood stream of the mammal for delivery to other sites within thebody.

To enhance the delivery of a drug through the skin, a modulating agentas described herein and a drug are contacted with the skin surface.Preferred modulating agents for use within such methods comprise acyclic peptide having an N-acetyl group, as described above, such asN-Ac-CHAVC-NH₂ (SEQ ID NO: 10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20),N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ ID NO:22),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30),N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ ID NO:36),N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48), aswell as derivatives thereof, including derivatives without the N-acetylgroup. Multifunctional modulating agents comprising the cadherin CARsequence HAV linked to one or more of the Dsc and/or the Dsg CARsequences may also be used to disrupt epithelial cell adhesion. Suchmodulating agents may also, or alternatively, comprise the fibronectinCAR sequence RGD, which is recognized by integrins, the occludin CARsequence LYHY (SEQ ID NO:55) and/or a claudin CAR sequences as describedabove. Alternatively, a separate modulator of non-classicalcadherin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately.

Contact may be achieved by direct application of the modulating agent,generally within a composition formulated as a cream or gel, or usingany of a variety of skin contact devices for transdermal application(such as those described in European Patent Application No. 566,816 A;U.S. Pat. No. 5,613,958; U.S. Pat. No. 5,505,956). A skin patch providesa convenient method of administration (particularly for slow-releaseformulations). Such patches may contain a reservoir of modulating agentand drug separated from the skin by a membrane through which the drugdiffuses. Within other patch designs, the modulating agent and drug maybe dissolved or suspended in a polymer or adhesive matrix that is thenplaced in direct contact with the patient's skin. The modulating agentand drug may then diffuse from the matrix into the skin. Modulatingagent(s) and drug(s) may be contained within the same composition orskin patch, or may be separately administered, although administrationat the same time and site is preferred. In general, the amount ofmodulating agent administered via the skin varies with the nature of thecondition to be treated or prevented, but may vary as described above.Such levels may be achieved by appropriate adjustments to the deviceused, or by applying a cream formulated as described above. Transfer ofthe drug across the skin and to the target tissue may be predicted basedon in vitro studies using, for example, a Franz cell apparatus, andevaluated in vivo by appropriate means hat will be apparent to those ofordinary skill in the art. As an example, monitoring of he serum levelof the administered drug over time provides a convenient measure of thedrug transfer across the skin.

Transdermal drug delivery as described herein is particularly useful insituations in which a constant rate of drug delivery is desired, toavoid fluctuating blood levels of a drug. For example, morphine is ananalgesic commonly used immediately following surgery. When givenintermittently in a parenteral form (intramuscular, intravenous), thepatient usually feels sleepy during the first hour, is well during thenext 2 hours and is in pain during the last hour because the blood levelgoes up quickly after the injection and goes down below the desirablelevel before the 4 hour interval prescribed for re-injection is reached.Transdermal administration as described herein permits the maintenanceof constant levels for long periods of time (e.g., days), which allowsadequate pain control and mental alertness at the same time. Insulinprovides another such example. Many diabetic patients need to maintain aconstant baseline level of insulin which is different from their needsat the time of meals. The baseline level may be maintained usingtransdermal administration of insulin, as described herein. Antibioticsmay also be administered at a constant rate, maintaining adequatebactericidal blood levels, while avoiding the high levels that are oftenresponsible for the toxicity (e.g., levels of gentamycin that are toohigh typically result in renal toxicity).

Drug delivery by the methods of the present invention also provide amore convenient method of drug administration. For example, it is oftenparticularly difficult to administer parenteral drugs to newborns andinfants because of the difficulty associated with finding veins ofacceptable caliber to catheterize. However, newborns and infants oftenhave a relatively large skin surface as compared to adults.

Transdermal drug delivery permits easier management of such patients andallows certain types of care that can presently be given only inhospitals to be given at home.

Other patients who typically have similar difficulties with venouscatheterization are patients undergoing chemotherapy or patients ondialysis. In addition, for patients undergoing prolonged therapy,transdermal administration as described herein is more convenient thanparenteral administration.

Transdermal administration as described herein also allows thegastrointestinal tract to be bypassed in situations where parenteraluses would not be practical. For example, there is a growing need formethods suitable for administration of therapeutic small peptides andproteins, which are typically digested within the gastrointestinaltract. The methods described herein permit administration of suchcompounds and allow easy administration over long periods of time.Patients who have problems with absorption through theirgastrointestinal tract because of prolonged ileus or specificgastrointestinal diseases limiting drug absorption may also benefit fromdrugs formulated for transdermal application as described herein.

Further, there are many clinical situations where it is difficult tomaintain compliance. For example, patients with mental problems (e.g.,patients with Alzheimer's disease or psychosis) are easier to manage ifa constant delivery rate of drug is provided without having to rely ontheir ability to take their medication at specific times of the day.Also patients who simply forget to take their drugs as prescribed areless likely to do so if they merely have to put on a skin patchperiodically (e.g., every 3 days). Patients with diseases that arewithout symptoms, like patients with hypertension, are especially atrisk of forgetting to take their medication as prescribed.

For patients taking multiple drugs, devices for transdermal applicationsuch as skin patches may be formulated with combinations of drugs thatare frequently used together. For example, many heart failure patientsare given digoxin in combination with furosemide. The combination ofboth drugs into a single skin patch facilitates administration, reducesthe risk of errors (taking the correct pills at the appropriate time isoften confusing to older people), reduces the psychological strain oftaking “so many pills,” reduces skipped dosage because of irregularactivities and improves compliance.

The methods described herein are particularly applicable to humans, butalso have a variety of veterinary uses, such as the administration ofgrowth factors or hormones (e.g., for fertility control) to an animal.

As noted above, a wide variety of drugs may be administered according tothe methods provided herein. Some examples of drug categories that maybe administered transdermally include anti-inflammatory drugs (e.g., inarthritis and in other condition) such as all NSAID, indomethacin,prednisone, etc.; analgesics (especially when oral absorption is notpossible, such as after surgery, and when parenteral administration isnot convenient or desirable), including morphine, codeine, Demerol,acetaminophen and combinations of these (e.g., codeine plusacetaminophen); antibiotics such as Vancomycin (which is not absorbed bythe GI tract and is frequently given intravenously) or a combination ofINH and Rifampicin (e.g., for tuberculosis); anticoagulants such asheparin (which is not well absorbed by the GI tract and is generallygiven parenterally, resulting in fluctuation in the blood levels with anincreased risk of bleeding at high levels and risks of inefficacy atlower levels) and Warfarin (which is absorbed by the GI tract but cannotbe administered immediately after abdominal surgery because of thenormal ileus following the procedure); antidepressants (e.g., insituations where compliance is an issue as in Alzheimer's disease orwhen maintaining stable blood levels results in a significant reductionof anti-cholinergic side effects and better tolerance by patients), suchas amitriptylin, imipramin, prozac, etc.; antihypertensive drugs (e.g.,to improve compliance and reduce side effects associated withfluctuating blood levels), such as diuretics and beta-blockers (whichcan be administered by the same patch; e.g., furosemide and propanolol);antipsychotics (e.g., to facilitate compliance and make it easier forcare giver and family members to make sure that the drug is received),such as haloperidol and chlorpromazine; and anxiolytics or sedatives(e.g., to avoid the reduction of alertness related to high blood levelsafter oral administration and allow a continual benefit throughout theday by maintaining therapeutic levels constant).

Numerous other drugs may be administered as described herein, includingnaturally occurring and synthetic hormones, growth factors, proteins andpeptides. For example, insulin and human growth hormone, growth factorslike erythropoietin, interleukins and interferons may be delivered viathe skin.

Kits for administering a drug via the skin of a mammal are also providedwithin the present invention. Such kits generally comprise a device fortransdermal application (i.e., skin patch) in combination with, orimpregnated with, one or more modulating agents. A drug may additionallybe included within such kits.

Within a related embodiment, the use of modulating agents as describedherein to increase skin permeability may also facilitate sampling of theblood compartment by passive diffision, permitting detection and/ormeasurement of the levels of specific molecules circulating in theblood. For example, application of one or more modulating agents to theskin, via a skin patch as described herein, permits the patch tofunction like a sponge to accumulate a small quantity of fluidcontaining a representative sample of the serum. The patch is thenremoved after a specified amount of time and analyzed by suitabletechniques for the compound of interest (e.g., a medication, hormone,growth factor, metabolite or marker). Alternatively, a patch may beimpregnated with reagents to permit a color change if a specificsubstance (e.g., an enzyme) is detected. Substances that can be detectedin this manner include, but are not limited to, illegal drugs such ascocaine, HIV enzymes, glucose and PSA. This technology is of particularbenefit for home testing kits.

Within a further aspect, methods are provided for enhancing delivery ofa drug to a tumor in a mammal, comprising administering a modulatingagent in combination with a drug to a tumor-bearing mammal. Modulatingagents for use within such methods include those designed to disruptE-cadherin and/or N-cadherin mediated cell adhesion, and comprise acyclic peptide such as N-Ac-CHAVC-NH₂ (SEQ ID 25 NO:8), N-Ac-CHAVDC-NH₂(SEQ ID NO:48), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:70), N-Ac-CHAVDINC-NH₂ (SEQID NO:71), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:72), N-Ac-CAHAVC-NH₂ (SEQ IDNO:49), N-Ac-CAHAVDC-NH₂ (SEQ ID NO: 16), N-Ac-CAHAVDIC-NH₂ (SEQ IDNO:10), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:50), N-Ac-CLRAHAVC-NH₂ (SEQ IDNO:51), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:52), N-Ac-CSHAVC-NH₂ (SEQ IDNO:12), N-Ac-CHAVSC-NH₂ (SEQ ID NO:14), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:53),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:18), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:54),N-Ac-KHAVD-NH₂ (SEQ ID NO:20), N-Ac-DHAVK-NH₂ (SEQ ID NO:55),N-Ac-KHAVE-NH₂ (SEQ ID NO:56), N-Ac-AHAVDI-NH₂ (SEQ ID NO:44),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:57), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:45) andderivatives thereof, including derivatives without the N-acetyl group.Bi-functional modulating agents that comprise an HAV sequence withflanking E-cadherin-specific sequences and an HAV sequence with flankingN-cadherin-specific sequences are also preferred.

In one particularly preferred embodiment, a modulating agent is capableof disrupting cell adhesion mediated by multiple adhesion molecules. Forexample, a single branched modulating agent (or multiple agents linkedto a single molecule or support material) may disrupt E-cadherin,N-cadherin, occludin, Dsc and Dsg mediated cell adhesion, therebydisrupting adherens junctions, tight junctions and desmosomes. Such anagent may comprise the cadherin CAR sequence, HAV, as well as one ormore of the fibronectin CAR sequence RGD, which is recognized byintegrins; a dsg CAR sequence; a dsc CAR sequence; a claudin CARsequence; an occludin CAR sequence and/or an OB-cadherin CAR sequence.Such agents serve as multifunctional disrupters of cell adhesion.Alternatively, a separate modulator of non-classical cadherin-mediatedcell adhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. Antibodies or Fab fragments directed against a cadherin CARsequence and/or an occludin CAR sequence may also be employed, eitherincorporated into a modulating agent or within a separate modulator thatis administered concurrently.

Preferably, the cyclic peptide and the drug are formulated within thesame composition or drug delivery device prior to administration. Ingeneral, a cyclic peptide may enhance drug delivery to any tumor, andthe method of administration may be chosen based on the type of targettumor. For example, injection or topical administration as describedabove may be preferred for melanomas and other accessible tumors (e.g.,metastases from primary ovarian tumors may be treated by flushing theperitoneal cavity with the composition). Other tumors (e.g., bladdertumors) may be treated by injection of the cyclic peptide and the drug(such as mitomycin C) into the site of the tumor. In other instances,the composition may be administered systemically, and targeted to thetumor using any of a variety of specific targeting agents. Suitabledrugs may be identified by those of ordinary skill in the art based uponthe type of cancer to be treated (e.g., mitomycin C for bladder cancer).In general, the amount of cyclic peptide administered varies with themethod of administration and the nature of the tumor, within the typicalranges provided above, preferably ranging from about 1 μg/mL to about 2mg/mL, and more preferably from about 10 μg/mL to 100 μg/mL. Transfer ofthe drug to the target tumor may be evaluated by appropriate means thatwill be apparent to those of ordinary skill in the art, such as areduction in tumor size. Drugs may also be labeled (e.g., usingradionuclides) to permit direct observation of transfer to the targettumor using standard imaging techniques.

Within a related aspect, the present invention provides methods forinhibiting the development of a cancer (i.e., for treating or preventingcancer and/or inhibiting metastasis) in a mammal. Cancer tumors aresolid masses of cells, growing out of control, which require nourishmentvia blood vessels. The formation of new capillaries is a prerequisitefor tumor growth and the emergence of metastases. Administration of amodulating agent as described herein may disrupt the growth of suchblood vessels, thereby providing effective therapy for the cancer and/orinhibiting metastasis. Modulating agents comprising cyclic peptides mayalso be used to treat leukemias. Preferred modulating agents for usewithin such methods include those that disrupt N-cadherin mediated celladhesion, and comprise a cyclic peptide such as N-Ac-CHAVC-NH₂ (SEQ IDNO:10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50),N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76),N-Ac-CAHAVC-NH₂ (SEQ ID NO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26),N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28),N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32),N-Ac-CSHAVC-NH₂ (SEQ ID NO:36), N-Ac-CH AVSC-NH₂ (SEQ ID NO:38),N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40), N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42),N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44), N-Ac-KHAVD-NH₂ (SEQ ID NO:12),N-Ac-DHAVK-NH₂ (SEQ ID NO:14), N-Ac-KHAVE-NH₂ (SEQ ID NO:16),N-Ac-AHAVDI-NH₂ (SEQ ID NO:34), N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77),N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) and derivatives thereof, includingderivatives without the N-acetyl group. In addition, a modulating agentmay comprise the sequence RGD, which is recognized by integrins, and/orthe occludin CAR sequence LYHY (SEQ ID NO:55) separated from the HAVsequence via a linker. Other CAR sequences that may be present includean OB-cadherin CAR sequence; dsc CAR sequence. dsg CAR sequence and/orclaudin CAR sequence. Alternatively, a separate modulator of integrin-OB-cadherin-, dsc-, dsg-, claudin- and/or occludin-mediated celladhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately.

A modulating agent may be administered alone (e.g., via the skin) orwithin a pharmaceutical composition. For melanomas and certain otheraccessible tumors, injection or topical administration as describedabove may be preferred. For ovarian cancers, flushing the peritonealcavity with a composition comprising one or more modulating agents mayprevent metastasis of ovarian tumor cells. Other tumors (e.g., bladdertumors, bronchial tumors or tracheal tumors) may be treated by injectionof the modulating agent into the cavity. In other instances, thecomposition may be administered systemically, and targeted to the tumorusing any of a variety of specific targeting agents, as described above.In general, the amount of modulating agent administered varies dependingupon the method of administration and the nature of the cancer, but mayvary within the ranges identified above. The effectiveness of the cancertreatment or inhibition of metastasis may be evaluated using well knownclinical observations such as the level of serum markers (e.g., CEA orPSA).

Within a further related aspect, a modulating agent may be used toinhibit angiogenesis (i.e., the growth of blood vessels frompre-existing blood vessels) in a mammal. In general, inhibition ofangiogenesis may be beneficial in patients afflicted with diseases suchas cancer or arthritis. Preferred modulating agents for inhibition ofangiogenesis include those comprising one or more of H-CHAVC-NH₂ (SEQ IDNO:10) N-Ac-CHAVDC-NH₂ (SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50),N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76),N-Ac-CAHAVC-NH₂ (SEQ ID NO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26),N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24), N-Ac-CRAHAVDC-NH2 (SEQ ID NO:28),N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO: 14),N-Ac-KHAVE-NH₂ (SEQ ID NO: 16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34) andderivatives thereof, including derivatives without the N-acetyl group.In addition, a modulating agent for use in inhibiting angiogenesis maycomprise the sequence RGD, which is recognized by integrins, theoccludin CAR sequence LYHY (SEQ ID NO:55) and/or a claudin CAR sequence,separated from the HAV sequence via a linker. Alternatively, a separatemodulator of integrin- and/or occludin-mediated cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately.

The effect of a particular modulating agent on angiogenesis maygenerally be determined by evaluating the effect of the peptide on bloodvessel formation. Such a determination may generally be performed, forexample, using a chick chorioallantoic membrane assay (Iruela-Arispe etal., Molecular Biology of the Cell 6:327-343, 1995). Briefly, amodulating agent may be embedded in a mesh composed of vitrogen at oneor more concentrations (e.g., ranging from about 1 to 100 μg/mesh). Themesh(es) may then be applied to chick chorioallantoic membranes. After24 hours, the effect of the peptide may be determined using computerassisted morphometric analysis. A modulating agent should inhibitangiogenesis by at least 25% at a concentration of 33 μg/mesh.

The addition of a targeting agent may be beneficial, particularly whenthe administration is systemic. Suitable modes of administration anddosages depend upon the condition to be prevented or treated but, ingeneral, administration by injection is appropriate. Dosages may vary asdescribed above. The effectiveness of the inhibition may be evaluatedgrossly by assessing the inability of the tumor to maintain growth andmicroscopically by an absence of nerves at the periphery of the tumor.

In yet another related aspect, the present invention provides methodsfor inducing apoptosis in a cadherin-expressing cell. In general,patients afflicted with cancer may benefit from such treatment.Preferred modulating agents for use within such methods comprise acyclic peptide such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂(SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQID NO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ IDNO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ IDNO:24), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ IDNO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂(SEQ IDNO:36),N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) andderivatives thereof, including derivatives without the N-acetyl group.Modulating agents comprising a CAR sequence for a second adhesionmolecule (e.g., RGD, LYHY (SEQ ID NO:55) or a CAR sequence forOB-cadherin, a desmoglein, a desmocollin or claudin) are also preferred.Alternatively, a separate modulator of cell adhesion mediated by anadhesion molecule that is not a cadherin may be administered inconjunction with the modulating agent(s), either within the samepharmaceutical composition or separately. Administration may be topical,via injection or by other means, and the addition of a targeting agentmay be beneficial, particularly when the administration is systemic.Suitable modes of administration and dosages depend upon the locationand nature of the cells for which induction of apoptosis is desired but,in general, dosages may vary as described above. A biopsy may beperformed to evaluate the level of induction of apoptosis.

The present invention also provides methods for enhancing drug deliveryto the central nervous system of a mammal. The blood/brain barrier islargely impermeable to most neuroactive agents, and delivery of drugs tothe brain of a mammal often requires invasive procedures. Using amodulating agent as described herein, however, delivery may be by, forexample, systemic administration of a cyclic peptide-drug-targetingagent combination, injection of a cyclic peptide (alone or incombination with a drug and/or targeting agent) into the carotid arteryor application of a skin patch comprising a modulating agent to the headof the patient. Certain preferred cyclic peptides for use within suchmethods are relatively small (e.g., a ring size of 4-10 residues;preferably 5-7 residues) and include peptides comprising a 5-residuering such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10) and N-Ac-KHAVD-NH₂ (SEQ IDNO:12). Other preferred modulating agents comprise a cyclic peptide suchas N-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20),N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ ID NO:22),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30),N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ ID NO:36),N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-N H₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK- NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) andderivatives thereof, including derivatives without the N-acetyl group.Also preferred are bi-functional modulating agents comprising a cadherinCAR sequence, along with an occludin CAR sequence LYHY (SEQ ID NO:55)and/or claudin CAR sequence, preferably joined by a linker.Alternatively, a separate modulator of occludin-mediated cell adhesionmay be administered in conjunction with the modulating agent(s), eitherwithin the same pharmaceutical composition or separately. Modulatingagents may further comprise antibodies or Fab fragments directed againstthe N-cadherin CAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:75). Fabfragments directed against the occludin CAR sequenceGVNPTAQSSGSLYGSQIYALCNQFYTPAAT-GLYVDQYLYHYCVVDPQE (SEQ ID NO:78) mayalso be employed, either incorporated into the modulating agent oradministered concurrently as a separate modulator.

In general, the amount of cyclic peptide administered varies with themethod of administration and the nature of the condition to be treatedor prevented, but typically varies as described above. Transfer of thedrug to the central nervous system may be evaluated by appropriate meansthat will be apparent to those of ordinary skill in the art, such asmagnetic resonance imaging (MRI) or PET scan (positron emittedtomography).

In still further aspects, the present invention provides methods forenhancing adhesion of cadherin-expressing cells. Within certainembodiments, a modulating agent may be linked to a support molecule orto a solid support as described above, resulting in a matrix thatcomprises multiple modulating agents. Within one such embodiment, thesupport is a polymeric matrix to which modulating agents and moleculescomprising other CAR sequencers) are attached (e.g., modulating agentsand molecules comprising RGD, LYHY (SEQ ID NO:55) or a CAR sequence forOB-cadherin, a desmoglein, a desmocollin or claudin, may be attached tothe same matrix, preferably in an alternating pattern). Such matricesmay be used in contexts in which it is desirable to enhance adhesionmediated by multiple cell adhesion molecules. Alternatively, themodulating agent itself may comprise multiple HAV sequences orantibodies (or fragments thereof), separated by linkers as describedabove. Either way, the modulating agent(s) function as a “biologicalglue” to bind multiple cadherin-expressing cells within a variety ofcontexts.

Within one embodiment, such modulating agents may be used to enhancewound healing and/or reduce scar tissue in a mammal. Preferredmodulating agents for use within such methods comprise a cyclic peptidesuch as N-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20),N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ ID NO:22),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30),N-Ac-CLRAHAVDC-H₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ ID NO:36),N-Ac-CHAVSC-NH₂ SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH2 SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) andderivatives thereof, including derivatives without the N-acetyl group.Modulating agents that are linked to a biocompatible and biodegradablematrix such as cellulose or collagen are particularly preferred. For usewithin such methods, a modulating agent should have a free amino orhydroxyl group. Multi-functional modulating agents comprising thecadherin CAR sequence, HAV, the fibronectin CAR sequence RGD, which isrecognized by integrins, as well CAR sequences for OB-cadherin, claudin,dsc or dsg, may also be used as potent stimulators of wound healingand/or to reduce scar tissue. Such agents may also, or alternatively,comprise the occludin CAR sequence LYHY (SEQ ID NO:55). Alternatively,one or more separate modulators of integrin-, Dsc-, Dsg-, claudin-,OB-cadherin- and/or occludin-mediated cell adhesion may be administeredin conjunction with the modulating agent(s), either within the samepharmaceutical composition or separately.

The modulating agents are generally administered topically to the wound,where they may facilitate closure of the wound and may augment, or evenreplace, stitches. Similarly, administration of matrix-linked modulatingagents may facilitate cell adhesion in foreign tissue implants (e.g.,skin grafting and prosthetic implants) and may prolong the duration andusefulness of collagen injection. In general, the amount ofmatrix-linked cyclic peptide administered to a wound, graft or implantsite varies with the severity of the wound and/or the nature of thewound, graft, or implant, but may vary as discussed above.

Within another embodiment, one or more modulating agents may be linkedto the interior surface of a tissue culture plate or other cell culturesupport, such as for use in a bioreactor. Such linkage may be performedby any suitable technique, as described above. Modulating agents linkedin this fashion may generally be used to immobilize cadherin-expressingcells. For example, dishes or plates coated with one or more modulatingagents may be used to immobilize cadherin-expressing cells within avariety of assays and screens. Within bioreactors (i.e., systems forlarger scale production of cells or organoids), modulating agents maygenerally be used to improve cell attachment and stabilize cell growth.Modulating agents may also be used within bioreactors to support theformation and function of highly differentiated organoids derived, forexample, from dispersed populations of fetal mammalian cells.Bioreactors containing biomatrices of cyclic peptide(s) may also be usedto facilitate the production of specific proteins.

Modulating agents as described herein may be used within a variety ofbioreactor configurations. In general, a bioreactor is designed with aninterior surface area sufficient to support larger numbers of adherentcells. This surface area can be provided using membranes, tubes,microtiter wells, columns, hollow fibers, roller bottles, plates,dishes, beads or a combination thereof. A bioreactor may becompartmentalized. The support material within a bioreactor may be anysuitable material known in the art; preferably, the support materialdoes not dissolve or swell in water. Preferred support materialsinclude, but are not limited to, synthetic polymers such as acrylics,vinyls, polyethylene, polypropylene, polytetrafluoroethylene, nylons,polyurethanes, polyamides, polysulfones and poly(ethyleneterephthalate); ceramics; glass and silica.

Modulating agents may also be used, within other aspects of the presentinvention, to enhance and/or direct neurological growth. In one aspect,neurite outgrowth may be enhanced and/or directed by contacting a neuronwith one or more modulating agents. Preferred modulating agents for usewithin such methods are linked to a polymeric matrix or other supportand include those peptides without substantial flanking sequences, asdescribed above. In particularly preferred embodiments, the modulatingagent comprises a cyclic peptide such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10),N-Ac-CHAVDC-NH₂ (SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50),N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76),N-Ac-CAHAVC-NH₂ (SEQ ID NO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26),N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28),N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32),N-Ac-CSHAVC-NH₂ (SEQ ID NO:36), N-Ac-CHAVSC-NH₂ (SEQ ID NO:38),N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40), N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42),N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44), N-Ac-KHAVD-NH₂ (SEQ ID NO: 12),N-Ac-DHAVK-NH₂ (SEQ ID NO: 14), N-Ac-KHAVE-NH₂ (SEQ ID NO:16),N-Ac-AHAVDI-NH₂ (SEQ ID NO:34), N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77),N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) and derivatives thereof, includingderivatives without the N-acetyl group. In addition, a modulating agentcomprising RGD and/or YIGSR (SEQ ID NO:52), which are bound byintegrins, the cadherin CAR sequence HAV, and/or the N-CAM CAR sequenceKYSFNYDGSE (SEQ ID NO:53) may further facilitate neurite outgrowth.Other CAR sequences that may also, or alternatively, be included are CARsequences for cadherin-7, cadherin-8, cadherin-12, cadherin-14,cadherin-15, PB-cadherin, protocadherins and cadherin-related neuronalreceptors. Modulating agents comprising antibodies, or fragmentsthereof, may be used within this aspect of the present invention withoutthe use of linkers or support materials. Preferred antibody modulatingagents include Fab fragments directed against the N-cadherin CARsequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:75). Fab fragments directedagainst the N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:53) may also beemployed, either incorporated into the modulating agent or administeredconcurrently as a separate modulator.

The method of achieving contact and the amount of modulating agent usedwill depend upon the location of the neuron and the extent and nature ofthe outgrowth desired. For example, a neuron may be contacted (e.g., viaimplantation) with modulating agent(s) linked to a support material suchas a suture, fiber nerve guide or other prosthetic device such that theneurite outgrowth is directed along the support material. Alternatively,a tubular nerve guide may be employed, in which the lumen of the nerveguide contains a composition comprising the modulating agent(s). Invivo, such nerve guides or other supported modulating agents may beimplanted using well known techniques to, for example, facilitate thegrowth of severed neuronal connections and/or to treat spinal cordinjuries. It will be apparent to those of ordinary skill in the art thatthe structure and composition of the support should be appropriate forthe particular injury being treated. In vitro, a polymeric matrix maysimilarly be used to direct the growth of neurons onto patternedsurfaces as described, for example, in U.S. Pat. No. 5,510,628.

Within another such aspect, one or more cyclic peptides may be used fortherapy of a demyelinating neurological disease in a mammal. There are anumber of demyelinating diseases, such as multiple sclerosis,characterized by oligodendrocyte death. It has been found, within thecontext of the present invention, that Schwann cell migration onastrocytes is inhibited by N-cadherin. Modulating agents that disruptN-cadherin mediated cell adhesion as described herein may be implantedinto the central nervous system with cells capable of replenishing anoligodendrocyte population. such as Schwann cells, oligodendrocytes oroligodendrocyte precursor cells. Such therapy may facilitate of the cellcapable of replenishing an oligodendrocyte population and permit thepractice of Schwann cell or oligodendrocyte replacement therapy.

Multiple sclerosis patients suitable for treatment may be identified bycriteria that establish a diagnosis of clinically definite or clinicallyprobable MS (see Poser et al., Ann. Neurol. 13:227, 1983). Candidatepatients for preventive therapy may be identified by the presence ofgenetic factors, such as HLA-type DR2a and DR2b, or by the presence ofearly disease of the relapsing remitting type.

Schwann cell grafts may be implanted directly into the brain along withthe modulating agent(s) using standard techniques. Preferred modulatingagents for use within such methods comprise a cyclic peptide such asN-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20),N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ ID NO:22),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30),N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ ID NO:36),N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) andderivatives thereof (including derivatives without the N-acetyl group).Modulating agents comprising antibodies, or fragments thereof, may alsobe used within this aspect of the present invention. Preferred antibodymodulating agents include Fab fragments directed against the N-cadherinCAR sequence FHLRAHAVDINGNQV-NH₂ (SEQ ID NO:75). Suitable amounts ofcyclic peptide generally range as described above, preferably from about10 μg/mL to about 1 mg/mL.

Alternatively, a modulating agent may be implanted with oligodendrocyteprogenitor cells (OPs) derived from donors not afflicted with thedemyelinating disease. The myelinating cell of the CNS is theoligodendrocyte. Although mature oligodendrocytes and immature cells ofthe oligodendrocyte lineage, such as the oligodendrocyte type 2astrocyte progenitor, have been used for transplantation, OPs are morewidely used. OPs are highly motile and are able to migrate fromtransplant sites to lesioned areas where they differentiate into maturemyelin-forming oligodendrocytes and contribute to repair of demyelinatedaxons (see e.g., Groves et al., Nature 362:453-55, 1993; Baron-VanEvercooren et al., Glia 16:147-64, 1996). OPs can be isolated usingroutine techniques known in the art (see e.g., Milner andFrench-Constant, Development 120:3497-3506, 1994), from many regions ofthe CNS including brain, cerebellum, spinal cord, optic nerve andolfactory bulb. Substantially greater yields of OP's are obtained fromembryonic or neonatal rather than adult tissue. OPs may be isolated fromhuman embryonic spinal cord and cultures of neurospheres established.Human fetal tissue is a potential valuable and renewable source of donorOP's for future, long range transplantation therapies of demyelinatingdiseases such as MS.

OPs can be expanded in vitro if cultured as “homotypic aggregates” or“spheres” (Avellana-Adalid et al, J. Neurosci. Res. 45:558-70, 1996).Spheres (sometimes called “oligospheres” or “neurospheres”) are formedwhen OPs are grown in suspension in the presence of growth factors suchas PDGF and FGF. OPs can be harvested from spheres by mechanicaldissociation and used for subsequent transplantation or establishment ofnew spheres in culture. Alternatively, the spheres themselves may betransplanted, providing a “focal reservoir” of OPs (Avellana-Adalid etal, J. Neurosci. Res. 45:558-70, 1996).

An alternative source of OP may be spheres derived from CNS stem cells.Recently, Reynolds and Weiss, Dev. Biol. 165:1-13, 1996 have describedspheres formed from EGF-responsive cells derived from embryonicneuroepithelium, which appear to retain the pluripotentiality exhibitedby neuroepithelium in vivo. Cells dissociated from these spheres areable to differentiate into neurons, oligodendrocytes and astrocytes whenplated on adhesive substrates in the absence of EGF, suggesting thatEGF-responsive cells derived from undifferentiated embryonicneuroepithelium may represent CNS stem cells (Reynolds and Weiss, Dev.Biol. 165:1-13, 1996). Spheres derived from CNS stem cells provide analternative source of OP which may be manipulated in vitro fortransplantation in vivo. Spheres composed of CNS stem cells may furtherprovide a microenvironment conducive to increased survival, migration,and differentiation of the OPs in vivo.

The use of neurospheres for the treatment of MS may be facilitated bymodulating agents that enhance cell migration from the spheres. In theabsence of modulating agent, the cells within the spheres adhere tightlyto one another and migration out of the spheres is hindered. Modulatingagents that disrupt N-cadherin mediated cell adhesion as describedherein, when injected with neurospheres into the central nervous system,may improve cell migration and increase the efficacy of OP replacementtherapy. Neurosphere grafts may be implanted directly into the centralnervous system along with the modulating agent(s) using standardtechniques.

Alternatively, a modulating agent may be administered alone or within apharmaceutical composition. The duration and frequency of administrationwill be determined by such factors as the condition of the patient, andthe type and severity of the patients disease. Within particularlypreferred embodiments of the invention, the cyclic peptide orpharmaceutical composition may be administered at a dosage ranging from0.1 mg/kg to 20 mg/kg, although appropriate dosages may be determined byclinical trials. Methods of administration include injection,intravenous or intrathecal (i.e., directly in cerebrospinal fluid).

Effective treatment of multiple sclerosis may be evidenced by any of thefollowing criteria: EDSS (extended disability status scale), appearanceof exacerbations or MRI (magnetic resonance imaging). The EDSS is ameans to grade clinical impairment due to MS (Kurtzke, Neurology33:1444, 1983), and a decrease of one full step defines an effectivetreatment in the context of the present invention (Kurtzke, Ann. Neurol.36:573-79, 1994). Exacerbations are defined as the appearance of a newsymptom that is attributable to MS and accompanied by an appropriate newneurologic abnormality (Sipe et al., Neurology 34:1368, 1984). Therapyis deemed to be effective if there is a statistically significantdifference in the rate or proportion of exacerbation-free patientsbetween the treated group and the placebo group or a statisticallysignificant difference in the time to first exacerbation or duration andseverity in the treated group compared to control group. MRI can be usedto measure active lesions using gadolinium-DTPA-enhanced imaging(McDonald et al. Ann. Neurol. 36:14, 1994) or the location and extent oflesions using T₂-weighted techniques. The presence, location and extentof MS lesions may be determined by radiologists using standardtechniques. Improvement due to therapy is established when there is astatistically significant improvement in an individual patient comparedto baseline or in a treated group versus a placebo group.

Efficacy of the modulating agent in the context of prevention may bejudged based on clinical measurements such as the relapse rate and EDSS.Other criteria include a change in area and volume of T2 images on MRI,and the number and volume of lesions determined by gadolinium enhancedimages.

Within a related aspect, the present invention provides methods forfacilitating migration of an N-cadherin expressing cell on astrocytes,comprising contacting an N-cadherin expressing cell with (a) a celladhesion modulating agent that inhibits cadherin-mediated cell adhesion,wherein the modulating agent comprises a cyclic peptide that comprisesthe sequence HAV; and (b) one or more astrocytes; and herebyfacilitating migration of the N-cadherin expressing cell on theastrocytes. Preferred N-cadherin expressing cells include Schwann cells,oligodendrocytes and oligodendrocyte progenitor cells.

Within another aspect, modulating agents as described herein may be usedfor modulating the immune system of a mammal in any of several ways.Cadherins are expressed on immature B and T cells (thymocytes and bonemarrow pre-B cells), as well as on specific subsets of activated B and Tlymphocytes and some hematological malignancies (see Lee et al., J.Immunol. 152:5653-5659, 1994; Munro et al., Cellular Immunol.169:309-312, 1996; Tsutsui et al., J. Biochem. 120:1034-1039, 1996;Cepek et al., Proc. Natl. Acad. Sci. USA 93:6567-6571, 1996). Modulatingagents may generally be used to modulate specific steps within cellularinteractions during an immune response or during the dissemination ofmalignant lymphocytes.

For example, a modulating agent as described herein may be used to treatdiseases associated with excessive generation of otherwise normal Tcells. Without wishing to be bound by any particular theory, it isbelieved that the interaction of cadherins on maturing T cells and Bcell subsets contributes to protection of these cells from programmedcell death. A modulating agent may decrease such interactions, leadingto the induction of programmed cell death. Accordingly, modulatingagents may be used to treat certain types of diabetes and rheumatoidarthritis, particularly in young children where the cadherin expressionon thymic pre-T cells is greatest.

Modulating agents may also be administered to patients afflicted withcertain skin disorders (such as cutaneous lymphomas), acute B cellleukemia and excessive immune reactions involving the humoral immunesystem and generation of immunoglobulins, such as allergic responses andantibody-mediated graft rejection. In addition, patients withcirculating cadherin-positive malignant cells (e.g., during regimeswhere chemotherapy or radiation therapy is eliminating a major portionof the malignant cells in bone marrow and other lymphoid tissue) maybenefit from treatment with a cyclic peptide. Such treatment may alsobenefit patients undergoing transplantation with peripheral blood stemcells.

Preferred modulating agents for use within such methods include thosethat disrupt E-cadherin and/or N-cadherin mediated cell adhesion, andcomprise a cyclic peptide such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10),N-Ac-CHAVDC-NH₂ (SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50),N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76),N-Ac-CAHAVC-NH₂ (SEQ ID NO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26),N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28),N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32),N-Ac-CSHAVC-NH₂ (SEQ ID NO:36), N-Ac-CHAVSC-NH₂ (SEQ ID NO:38),N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40), N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42),N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44), N-Ac-KHAVD-NH₂ (SEQ ID NO:12),N-Ac-DHAVK-NH₂ (SEQ ID NO:14), N-Ac-KHAVE-NH₂ (SEQ ID NO:16),N-Ac-AHAVDI-NH₂ (SEQ ID NO:34), N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77),N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) and derivatives thereof, includingderivatives without the N-acetyl group. In addition, a preferredmodulating agent may comprise one or more additional CAR sequences, suchas the sequence RGD, which is bound by integrins, as well as CARsequences for occludin, N-CAM, OB-cadherin, cadherin-5, cadherin-6 andcadherin-8. As noted above, such additional sequence(s) may be separatedfrom the HAV sequence via a linker. Alternatively, a separate modulatorof integrin-mediated cell adhesion may be administered in conjunctionwith the modulating agent(s), either within the same pharmaceuticalcomposition or separately.

Within the above methods, the modulating agent(s) are preferablyadministered systemically (usually by injection) or topically. A cyclicpeptide may be linked to a targeting agent. As noted above, a modulatingagent may further be linked to a targeting agent. For example, targetingto the bone marrow may be beneficial. A suitable dosage is sufficient toeffect a statistically significant reduction in the population of Band/or T cells that express cadherin and/or an improvement in theclinical manifestation of the disease being treated. Typical dosagesrange as described above.

Within further aspects, the present invention provides methods and kitsfor preventing pregnancy in a mammal. In general, disruption ofE-cadherin function prevents the adhesion of trophoblasts and theirsubsequent fusion to form syncitiotrophoblasts. In one embodiment, oneor more modulating agents as described herein may be incorporated intoany of a variety of well known contraceptive devices, such as spongessuitable for intravaginal insertion (see, e.g., U.S. Pat. No. 5,417,224)or capsules for subdermal implantation. Other modes of administrationare possible, however, including transdermal administration, formodulating agents linked to an appropriate targeting agent. Preferredmodulating agents for use within such methods comprise a cyclic peptidesuch as N-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂ (SEQ ID NO:20),N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ ID NO:22),N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24),N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30),N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ ID NO:36),N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48), andderivatives thereof, including derivatives without the N-acetyl group.In addition, a preferred modulating agent may comprise additional CARsequences, such as the sequence RGD, which is bound by integrins. Asnoted above, such additional sequences may be separated from the HAVsequence via a linker. Alternatively, a separate modulator ofintegrin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately.

Suitable methods for incorporation into a contraceptive device dependupon the type of device and are well known in the art. Such devicesfacilitate administration of the cyclic peptide(s) to the uterine regionand may provide a sustained release of the cyclic peptide(s). Ingeneral, cyclic peptide(s) may be administered via a contraceptivedevice at a dosage ranging from 0.1 to 20 mg/kg, although appropriatedosages may be determined by monitoring hCG levels in the urine. hCG isproduced by the placenta, and levels of this hormone rise in the urineof pregnant women. The urine hCG levels can be assessed byradio-immunoassay using well known techniques. Kits for preventingpregnancy generally comprise a contraceptive device impregnated with oneor more cyclic peptides.

Alternatively, a sustained release formulation of one or more cyclicpeptides may be implanted, typically subdermally, in a mammal for theprevention of pregnancy. Such implantation may be performed using wellknown techniques. Preferably, the implanted formulation provides adosage as described above, although the minimum effective dosage may bedetermined by those of ordinary skill in the art using, for example, anevaluation of hCG levels in the urine of women.

The present invention also provides methods for increasingvasopermeability in a mammal by administering one or more modulatingagents or pharmaceutical compositions. Within blood vessels, endothelialcell adhesion (mediated by N-cadherin) results in decreased vascularpermeability. Accordingly, modulating agents as described herein may beused to increase vascular permeability. Within certain embodiments,preferred modulating agents for use within such methods include peptidescapable of decreasing both endothelial and tumor cell adhesion. Suchmodulating agents may be used to facilitate the penetration ofanti-tumor therapeutic or diagnostic agents (e.g., monoclonalantibodies) through endothelial cell permeability barriers and tumorbarriers. Particularly preferred modulating agents comprise a cyclicpeptide such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10), N-Ac-CHAVDC-NH₂ (SEQ IDNO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (SEQ IDNO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76), N-Ac-CAHAVC-NH₂ (SEQ IDNO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26), N-Ac-CAHAVDIC-NH₂ (SEQ IDNO:24), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28), N-Ac-CLRAHAVC-NH₂ (SEQ IDNO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32), N-Ac-CSHAVC-NH₂ (SEQ IDNO:36), N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40),N-Ac-CSAVSSC-NH₂ (SEQ ID NO:42), N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44),N-Ac-KHAVD-NH₂ (SEQ ID NO:12), N-Ac-DHAVK-NH₂ (SEQ ID NO:14),N-Ac-KHAVE-NH₂ (SEQ ID NO:16), N-Ac-AHAVDI-NH₂ (SEQ ID NO:34),N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77), N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) andderivatives thereof, including derivatives without an N-acetyl group. Inaddition, a preferred modulating agent may comprise an occludin CARsequence LYHY (SEQ ID NO:55) and/or a CAR sequence for OB-cadherin orclaudin. As noted above, such an additional sequence may be separatedfrom the HAV sequence via a linker. Alternatively, a separate modulatorof occludin mediated cell adhesion may be administered in conjunctionwith one or modulating agents, either within the same pharmaceuticalcomposition or separately.

Within certain embodiments, preferred modulating agents for use withinsuch methods include cyclic peptides capable of decreasing bothendothelial and tumor cell adhesion. Such modulating agents may be usedto facilitate the penetration of anti-tumor therapeutic or diagnosticagents (e.g., monoclonal antibodies) through endothelial cellpermeability barriers and tumor barriers. For example, a modulatingagent may comprise an HAV sequence with flanking E-cadherin-specificsequences and an HAV sequence with flanking N-cadherin-specificsequences. Alternatively, separate modulating agents capable ofdisrupting N- and E-cadherin mediated adhesion may be administeredconcurrently.

In one particularly preferred embodiment, a modulating agent is furthercapable of disrupting cell adhesion mediated by multiple adhesionmolecules. Such an agent may comprise the cadherin CAR sequence, HAV, aswell as an RGD sequence, a Dsc CAR sequence, a Dsg CAR sequence and/orthe occludin CAR sequence LYHY (SEQ ID NO:55). Alternatively, a separatemodulator of non-classical cadherin-mediated cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately. Fab fragmentsdirected against any of the above CAR sequences may also be employed,either incorporated into a modulating agent or within a separatemodulator that is administered concurrently.

Treatment with a modulating agent may be appropriate, for example, priorto administration of an anti-tumor therapeutic or diagnostic agent(e.g., a monoclonal antibody or other macromolecule), an antimicrobialagent or an anti-inflammatory agent, in order to increase theconcentration of such agents in the vicinity of the target tumor,organism or inflammation without increasing the overall dose to thepatient. Modulating agents for use within such methods may be linked toa targeting agent to further increase the local concentration ofmodulating agent, although systemic administration of a vasoactive agenteven in the absence of a targeting agent increases the perfusion ofcertain tumors relative to other tissues. Suitable targeting agentsinclude antibodies and other molecules that specifically bind to tumorcells or to components of structurally abnormal blood vessels. Forexample, a targeting agent may be an antibody that binds to a fibrindegradation product or a cell enzyme such as a peroxidase that isreleased by granulocytes or other cells in necrotic or inflamed tissues.

Administration via intravenous injection or transdermal administrationis generally preferred. Effective dosages are generally sufficient toincrease localization of a subsequently administered diagnostic ortherapeutic agent to an extent that improves the clinical efficacy oftherapy of accuracy of diagnosis to a statistically significant degree.Comparison may be made between treated and untreated tumor host animalsto whom equivalent doses of the diagnostic or therapeutic agent areadministered. In general, dosages range as described above.

Within a further aspect, modulating agents as described herein may beused for controlled inhibition of synaptic stability, resulting inincreased synaptic plasticity. Within this aspect, administration of oneor more modulating agents may be advantageous for repair processeswithin the brain, as well as learning and memory, in which neuralplasticity is a key early event in the remodeling of synapses. Celladhesion molecules, particularly N-cadherin and E-cadherin, can functionto stabilize synapses, and loss of this function is thought to be theinitial step in the remodeling of the synapse that is associated withlearning and memory (Doherty et al., J. Neurobiology, 26:437-446, 1995;Martin and Kandel, Neuron, 17:567-570, 1996; Fannon and Colman, Neuron,17:423-434, 1996). Inhibition of cadherin function by administration ofone or more modulating agents that inhibit cadherin function maystimulate learning and memory.

Preferred modulating agents for use within such methods include thosethat disrupt E-cadherin and/or N-cadherin mediated cell adhesion, andcomprise cyclic peptides such as N-Ac-CHAVC-NH₂ (SEQ ID NO:10),N-Ac-CHAVDC-NH₂ (SEQ ID NO:20), N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50),N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51), N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76),N-Ac-CAHAVC-NH₂ (SEQ ID NO:22), N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26),N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24), N-Ac-CRAHAVDC-NH₂ (SEQ ID NO:28),N-Ac-CLRAHAVC-NH₂ (SEQ ID NO:30), N-Ac-CLRAHAVDC-NH₂ (SEQ ID NO:32),N-Ac-CSHAVC-NH₂ (SEQ ID NO:36), N-Ac-CHAVSC-NH₂ (SEQ ID NO:38),N-Ac-CSHAVSC-NH₂ (SEQ ID NO:40), N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42),N-Ac-CHAVSSC-NH₂ (SEQ ID NO:44), N-Ac-KHAVD-NH₂ (SEQ ID NO:12),N-Ac-DHAVK-NH₂ (SEQ ID NO:14), N-Ac-KHAVE-NH₂ (SEQ ID NO:16),N-Ac-AHAVDI-NH₂ (SEQ ID NO:34), N-Ac-SHAVDSS-NH₂ (SEQ ID NO:77),N-Ac-KSHAVSSD-NH₂ (SEQ ID NO:48) and derivatives thereof, includingderivatives without the N-acetyl group. In addition, a preferredmodulating agent may comprise one or more additional CAR sequences, suchas the sequence RGD, which is bound by integrins, the N-CAM CAR sequenceKYSFNYDGSE (SEQ ID NO:53) and/or a cadherin-related neuronal receptorCAR sequence. As noted above, such additional sequence(s) may beseparated from the HAV sequence via a linker. Alternatively, a separatemodulator of integrin and/or N-CAM mediated cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately. For such aspects,administration may be via encapsulation into a delivery vehicle such asa liposome, using standard techniques, and injection into, for example,the carotid artery. Alternatively, a modulating agent may be linked to adisrupter of the blood-brain barrier. In general dosages range asdescribed above.

Other aspects of the present invention provide methods that employantibodies raised against the modulating agents for diagnostic and assaypurposes. Such polyclonal and monoclonal antibodies may be raisedagainst a cyclic peptide using conventional techniques known to those ofordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988. In one suchtechnique, an immunogen comprising the cyclic peptide is initiallyinjected into any of a wide variety of mammals (e.g., mice, rats,rabbits, sheep or goats). Because of its small size, the cyclic peptideshould be joined to a carrier protein, such as bovine serum albumin orkeyhole limpet hemocyanin. Following one or more injections, the animalsare bled periodically. Polyclonal antibodies specific for the cyclicpeptide may then be purified from such antisera by, for example,affinity chromatography using the polypeptide coupled to a suitablesolid support.

Monoclonal antibodies specific for the cyclic peptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity from spleen cellsobtained from an animal immunized as described above. The spleen cellsare immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal.Single colonies are selected and their culture supernatants tested forbinding activity against the polypeptide. Hybridomas having highreactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation, and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target cadherin islocalized.

Cyclic peptides may also be used to generate monoclonal antibodies, asdescribed above, that are specific for particular cadherins (e.g.,antibodies that bind to E-cadherin, but do not bind significantly toN-cadherin, or vise versa). Such antibodies may generally be used fortherapeutic, diagnostic and assay purposes.

Assays typically involve using an antibody to detect the presence orabsence of a cadherin (free or on the surface of a cell), or proteolyticfragment containing the EC1 domain in a suitable biological sample, suchas tumor or normal tissue biopsies, blood, lymph node, serum or urinesamples, or other tissue, homogenate, or extract thereof obtained from apatient.

There are a variety of assay formats known to those of ordinary skill inthe art for using an antibody to detect a target molecule in a sample.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the assay may be performed in aWestern blot format, wherein a protein preparation from the biologicalsample is submitted to gel electrophoresis, transferred to a suitablemembrane and allowed to react with the antibody. The presence of theantibody on the membrane may then be detected using a suitable detectionreagent, as described below.

In another embodiment, the assay involves the use of antibodyimmobilized on a solid support to bind to the target cadherin, or aproteolytic fragment containing the EC1 domain, and remove it from theremainder of the sample. The bound cadherin may then be detected using asecond antibody or reagent that contains a reporter group.Alternatively, a competitive assay may be utilized, in which a cadherinis labeled with a reporter group and allowed to bind to the immobilizedantibody after incubation of the antibody with the sample. The extent towhich components of the sample inhibit the binding of the labeledcadherin to the antibody is indicative of the reactivity of the samplewith the immobilized antibody, and as a result, indicative of the levelof the cadherin in the sample.

The solid support may be any material known to those of ordinary skillin the art to which the antibody may be attached, such as a test well ina microtiter plate, a nitrocellulose filter or another suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic such as polystyrene orpolyvinylchloride. The antibody may be immobilized on the solid supportusing a variety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature.

In certain embodiments, the assay for detection of a cadherin in asample is a two-antibody sandwich assay. This assay may be performed byfirst contacting an antibody that has been immobilized on a solidsupport, commonly the well of a microtiter plate, with the biologicalsample, such that the cadherin within the sample is allowed to bind tothe immobilized antibody (a 30 minute incubation time at roomtemperature is generally sufficient). Unbound sample is then removedfrom the immobilized cadherin-antibody complexes and a second antibody(containing a reporter group such as an enzyme, dye, radionuclide,luminescent group, fluorescent group or biotin) capable of binding to adifferent site on the cadherin is added. The amount of second antibodythat remains bound to the solid support is then determined using amethod appropriate for the specific reporter group. The method employedfor detecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate.

Spectroscopic methods may be used to detect dyes, luminescent groups andfluorescent groups. Biotin may be detected using avidin, coupled to adifferent reporter group (commonly a radioactive or fluorescent group oran enzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.Standards and standard additions may be used to determine the level ofcadherin in a sample, using well known techniques.

The present invention also provides kits for use in such immunoassays.Such kits generally comprise one or more antibodies, as described above.In addition, one or more additional compartments or containers of a kitgenerally enclose elements, such as reagents, buffers and/or washsolutions, to be used in the immunoassay.

Within further aspects, cyclic peptides or antibodies thereto may beused to facilitate cell identification and sorting in vitro or imagingin vivo, permitting the selection of cells expressing differentcadherins (or different cadherin levels). Preferably, the cyclicpeptide(s) or antibodies for use in such methods are linked to adetectable marker. Suitable markers are well known in the art andinclude radionuclides, luminescent groups, fluorescent groups, enzymes,dyes, constant immunoglobulin domains and biotin. Within one preferredembodiment, a cyclic peptide or antibody linked to a fluorescent marker,such as fluorescein, is contacted with the cells, which are thenanalyzed by fluorescence activated cell sorting (FACS).

As noted above, in addition to diagnostic and assay purposes, antibodiesas described herein may be used in vitro or in vivo to modulate celladhesion. Within certain embodiments, antibodies may be used withinmethods in which enhanced cell adhesion is desired, as described above.For example, antibodies may be used within the above methods forenhancing and/or directing neurite outgrowth in vitro or in vivo.Antibodies may be used within the lumen of a tubular nerve guide or maybe attached to a fiber nerve guide, suture or other solid support andused as described above for cyclic peptides. Antibody dosages aresufficient to enhance or direct neurite outgrowth, and will vary withthe method of administration and the condition to be treated.

Antibodies may also be used as a “biological glue,” as described aboveto bind multiple cadherin-expressing cells within a variety of contexts,such as to enhance wound healing and/or reduce scar tissue, and/or tofacilitate cell adhesion in skin grafting or prosthetic implants. Ingeneral, the amount of matrix-linked antibody administered to a wound,graft or implant site varies with the severity of the wound and/or thenature of the wound, graft, or implant, but may vary as discussed above.Antibodies may also be linked to any of a variety of support materials,as described above, for use in tissue culture or bioreactors.

Within certain embodiments, antibodies (or, preferably, antigen-bindingfragments thereof) may be used in situations where inhibition of celladhesion is desired. Such antibodies or fragments may be used, forexample, for treatment of demyelinating diseases, such as MS, or toinhibit interactions between tumor cells, as described above. The use ofFab fragments is generally preferred.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Preparation of Representative Cyclic Peptides

This Example illustrates the solid phase synthesis of representativecyclic peptides.

The peptides were assembled on methylbenzhydrylamine resin (MBHA resin)for the C-terminal amide peptides. The traditional Merrifield resinswere used for any C-terminal acid peptides. Bags of a polypropylene meshmaterial were filled with the resin and soaked in dichloromethane. Theresin packets were washed three times with 5% diisopropylethylamine indichloromethane and then washed with dichloromethane. The packets arethen sorted and placed into a Nalgene bottle containing a solution ofthe amino acid of interest in dichloromethane. An equal amount ofdiisopropylcarbodiimide (DIC) in dichloromethane was added to activatethe coupling reaction. The bottle was shaken for one hour to ensurecompletion of the reaction. The reaction mixture was discarded and thepackets washed with DMF. The N-α-Boc was removed by acidolysis using a55% TFA in dichloromethane for 30 minutes leaving the TFA salt of theα-amino group. The bags were washed and the synthesis completed byrepeating the same procedure while substituting for the correspondingamino acid at the coupling step. Acetylation of the N-terminal wasperformed by reacting the peptide resins with a solution of aceticanhydride in dichloromethane in the presence of diisopropylethylamine.The peptide was then side-chain deprotected and cleaved from the resinat 0° C. with liquid HF in the presence of anisole as a carbocationscavenger.

The crude peptides were purified by reversed-phase high-performanceliquid chromatography. Purified linear precursors of the cyclic peptideswere solubilized in 75% acetic acid at a concentration of 2-10 mg/mL. A10% solution of iodine in methanol was added dropwise until a persistentcoloration was obtained. A 5% ascorbic acid solution in water was thenadded to the mixture until discoloration. The disulfide bridgecontaining compounds were then purified by HPLC and characterized byanalytical HPLC and by mass spectral analysis.

EXAMPLE 2 Disruption of the Ability of Mouse Cerebellar Neurons toExtend Neurites

Three cell adhesion molecules, N-cadherin, N-CAM and L1, are capable ofregulating neurite outgrowth (Doherty and Walsh, Curr. Op. Neurobiol.4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994; Hall et al.,Cell Adhesion and Commun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell.Neurosci. 8:99-111, 1994; Safell et al., Neuron 18:231-242, 1997).Neurons cultured on monolayers of 3T3 cells that have been transfectedwith cDNAs encoding N-cadherin, N-CAM or L1 extend longer neurites thanneurons cultured on 3T3 cells not expressing these cell adhesionmolecules. This Example illustrates the use of a representative cyclicpeptide to inhibit neurite outgrowth.

Neurons were cultured on monolayers of 3T3 cells transfected with cDNAencoding N-cadherin essentially as described by Doherty and Walsh, Curr.Op. Neurobiol. 4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994;Hall et al., Cell Adhesion and Commun. 3:441-450, 1996; Doherty andWalsh, Mol. Cell. Neurosci. 8:99-111, 1994; Safell et al., Neuron18:231-242, 1997. Briefly, monolayers of control 3T3 fibroblasts and 3T3fibroblasts that express N-cadherin were established by overnightculture of 80,000 cells in individual wells of an 8-chamber well tissueculture slide. 3000 cerebellar neurons isolated from post-natal day 3mouse brains were cultured for 18 hours on the various monolayers incontrol media (SATO/2% FCS), or media supplemented with variousconcentrations of the cyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10)or acontrol peptide without the HAV sequence (N-Ac-CHGVC-NH₂; SEQ ID NO:11).The cultures were then fixed and stained for GAP43 which specificallybinds to the neurons and their neurites. The length of the longestneurite on each GAP43 positive neuron was then measured by computerassisted morphometry.

As shown in FIG. 22, culture for 18 hours with N-Ac-CHAVC-NH₂ (SEQ IDNO:10) at a concentration of 500 μg/mL inhibited neurite outgrowth on3T3 cells expressing N-cadherin, whereas the cyclic peptideN-Ac-CHGVC-NH₂ (SEQ ID NO:11) (also at a concentration of 500 μg/ml) hadno effect on this process. Furthermore, the cyclic peptideN-Ac-CHAVC-NH₂ (SEQ ID NO:10) (used at a concentration of 500 μg/ml) didnot inhibit neurite outgrowth on 3T3 cells not expressing N-cadherin,N-CAM, or L1 (control cells), thus indicating that the peptide is nottoxic and that it has no non-specific effects on neurite outgrowth (FIG.22). These data also indicate that the peptide does not effect integrinfunction.

A dose-response study demonstrated that N-Ac-CHAVC-NH₂ (SEQ ID NO:10)significantly inhibited neurite outgrowth on 3T3 cells expressingN-cadherin at concentration of 50 μg/mL, and completely inhibitedneurite outgrowth on these cells at a concentration of 500 μg/mL (FIG.23). Finally, N-Ac-CHAVC-NH₂ (SEQ ID NO:10) (used at a concentration of500 μg/mL) did not inhibit neurite outgrowth on 3T3 cells expressingeither N-CAM or L1 (FIG. 4). These results indicate that the peptideN-Ac-CHAVC-NH₂ (SEQ ID NO:10) specifically inhibits the function ofN-cadherin. Collectively, the results obtained from these studiesdemonstrate that N-Ac-CHAVC-NH₂ (SEQ ID NO:10) is an effective inhibitorof neurite outgrowth by virtue of its ability to disrupt N-cadherinfunction.

EXAMPLE 3 Disruption of Bovine Endothelial Cell Adhesion

This Example illustrates the use of representative cyclic peptides todisrupt adhesion of endothelial cells, which express N-cadherin.

Bovine pulmonary artery endothelial cells were harvested by sterileablation and digestion in 0.1% collagenase (type II; WorthingtonEnzymes, Freehold, N.J.). Cells were maintained in Dulbecco's minimumessential medium (Clonetics, San Diego, Calif.) supplemented with 10%fetal calf serum (Atlantic Biologicals, Nor cross, Ga.) and 1%antibiotic-antimycotic at 37° C. in 7% CO₂ in air. Cultures werepassaged weekly in trypsin-EDTA (Gibco, Grand Island, N.Y.) and seededonto tissue culture plastic at 20,000 cells/m² for all experiments.Endothelial cultures were used at 1 week in culture, which isapproximately 3 days after culture confluency was established. The cellsused in all protocols were between 4th passage and 10th passage. Thecells were seeded onto coverslips and treated 30 minutes withN-Ac-CHAVC-NH₂ (SEQ ID NO:10) or N-Ac-CHGVC-NH₂ (SEQ ID NO:11) at 500μg/ml and then fixed with 1% paraformaldehyde.

The peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) disrupted the endothelial cellmonolayer within 30 minutes after being added to the culture medium,whereas N-Ac-CHGVC-NH₂ (SEQ ID NO:11) had no affect on the cells (FIG.5). Endothelial cell morphology was dramatically affected byN-Ac-CHAVC-NH₂ (SEQ ID NO:10), and the cells retracted from one anotherand became non-adherent. These data demonstrate that N-Ac-CHAVC-NH₂ (SEQID NO:10)is capable of inhibiting endothelial cell adhesion.

Under the same conditions, the cyclic peptides H-CHAVC-NH₂ (SEQ IDNO:10), N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24) (FIG. 6) and N-Ac-CHAVSC-NH₂(SEQ ID NO:38) had no effect on endothelial cell morphology, indicatingthat not all cyclic HAV-containing peptides are capable of disruptingendothelial cell adhesion at a concentration of 500 μg/mL. It is notunexpected that the potencies of individual cyclic peptides will varyThe cyclic peptide N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26; FIG. 7) had a slighteffect while N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42; FIG. 8) disrupted theendothelial cell monolayer and caused the cells to retract from oneanother.

EXAMPLE 4 Disruption of Human Ovarian Cancer Cell Adhesion

This Example illustrates the use of a representative cyclic peptide todisrupt adhesion of human ovarian cancer cells.

The human ovarian cancer cell line SKOV3 (ATCC #HTB-77) expressesN-cadherin. SKOV3 cells were cultured in a modified MEM-based mediacontaining 10% FCS. Cells were grown in T-250 culture flasks andmaintained by periodic subculturing. Cyclic peptides were tested oncells grown in individual wells of 96-well culture dishes (surface areaof each well was 0.32cm²). Cells were harvested from flasks and seededat a density of 50,000 cells per well in 0.1 mL media containing thecyclic peptides at concentrations of 1, 0.1, or 0.01 mg/mL, or in theabsence of cyclic peptide. Media control wells were also established.Cultures were evaluated periodically by microscopic examination underboth bright field and phase contrast conditions. Cultures weremaintained for 48 hours.

As shown in FIGS. 9A (compare to FIG. 9C) and 10A, the peptideN-Ac-CHAVC-NH₂ (SEQ ID NO:10) (final concentration of 1 mg/mL media)disrupted SKOV3 cell adhesion within 24 hours, whereas the controlN-Ac-CHGVC-NH₂ (SEQ ID NO:11) had no affect on cell adhesion (FIGS. 9Band 10B). The effect of different amounts of N-Ac-CHAVC-NH₂ (SEQ IDNO:10) after 48 hours is shown in FIGS. 9D-F. In the presence ofN-Ac-CHGVC-NH₂, (SEQ ID NO:11) (FIGS. 9B and 10B) the SKOV3 cells formedtightly adherent monolayers. In contrast, the SKOV3 cells did not spreadonto the substrata, nor did they form tightly adherent monolayers in thepresence of N-Ac-CHAVC-NH₂ (SEQ ID NO:10; FIGS. 9A, 9D and 10A). Thesedata demonstrate that N-Ac-CHAVC-NH₂ (SEQ ID NO:10) is capable ofinhibiting the function of human N-cadherin.

The cyclic peptides N-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24), N-Ac-CAHAVDC-NH₂(SEQ ID NO:26) and N-Ac-KHAVD-NH₂ (SEQ ID NO:12) were inactive in theSKOV3 cells, indicating that not all cyclic HAV-containing peptides arecapable of disrupting epithelial cell adhesion at concentrations of0.01-1 mg/mL It is not unexpected that the potencies of the cyclicpeptides will vary.

EXAMPLE 5 Disruption of Angiogenesis

Blood vessels are composed of adherent endothelial cells. This Exampleillustrates the use of a representative cyclic peptide to blockangiogenesis (the growth of blood vessels from pre-existing bloodvessels).

The chick chorioallantoic membrane assay was used to assess the effectsof cyclic peptides on angiogenesis (Iruela-Arispe et al., MolecularBiology of the Cell 6:327-343, 1995). Cyclic peptides were embedded in amesh composed of vitrogen at concentrations of 3, 17, and 33 μg/mesh.The meshes were then applied to 12-day-old chick embryonicchorioallantoic membranes. After 24 hours, the effects of the peptideson angiogenesis were assessed by computer assisted morphometricanalysis.

The ability of representative cyclic peptides to inhibit angiogenesis isillustrated by the results presented in Table 2. For each concentrationof cyclic peptide, the percent inhibition of angiogenesis (relative tothe level of angiogenesis in the absence of cyclic peptide) is provided.Assays were performed in the presence (+) or absence (−) of 0.01 mMVEGF. For example, the cyclic peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10)inhibited angiogenesis by 46%, 51%, and 51% at concentrations of 3, 17,and 33 μg/mesh, respectively. The N-cadherin selective peptidesN-Ac-CAHAVDIC-NH₂ (SEQ ID NO:24) and N-Ac-CAHAVDC-NH₂ (SEQ ID NO:26)also inhibited angiogenesis significantly. The E-cadherin selectivecyclic peptides N-Ac-CHAVSC-NH₂ (SEQ ID NO:38) and N-Ac-CSHAVSSC-NH₂(SEQ ID NO:42), as well as the scrambled peptide N-Ac-CVAHC-NH₂ (SEQ IDNO:18), were found to be relatively inactive in this assay.

TABLE 2 Concentration, μg/mesh ± VEGF Compound 3(−) 3(+) 17(−) 17(+)33(−) 33(+) H-CHAVC-NH₂ 11% 27% 13% 34% 17% 35% (SEQ ID NO: 10)N-Ac-CHAVSC-NH₂ 11% 17% 12% 16% 17% 19% (SEQ ID NO: 38) N-Ac-CVAHC-NH₂−1%  7% 13% 24% 12% 25% (SEQ ID NO: 18) N-Ac-CHAVC-NH₂ 12% 46% 22% 51%28% 51% (SEQ ID NO: 10) N-Ac-CAHAVDIC-NH₂ −1% 21% 15% 37% 33% 49% (SEQID NO: 24) N-Ac-CAHAVDC-NH₂ 21% 59% 27% 72% 31% 79% (SEQ ID NO: 26)N-Ac-CSHAVSSC-NH₂  1% −3% −3% 12% 17%  7% (SEQ ID NO: 42)

EXAMPLE 6 Disruption of Normal Rat Kidney (NRK Cell Adhesion

NRK cells express E-cadherin, and monolayer cultures of these cellsexhibit a cobblestone morphology. This Example illustrates the abilityof a representative cyclic peptide to disrupt NRK cell adhesion.

NRK cells (ATCC #1571-CRL) were plated at 10-20,000 cells per 35 mmtissue culture flasks containing DMEM with 10% FCS and sub-culturedperiodically (Laird et al., J. Cell Biol. 131:1193-1203, 1995). Cellswere harvested and replated in 35 mm tissue culture flasks containing 1mm coverslips and incubated until 5065% confluent (24-36 hours). At thistime, coverslips were transferred to a 24-well plate, washed once withfresh DMEM and exposed to cyclic peptide solutions (N-Ac-CHAVC-NH₂ (SEQID NO: 10) and N-Ac-CHGVC-NH₂ (SEQ ID NO:11)) at a concentration of 1mg/mL for 24 hours. Fresh peptide solutions were then added and thecells were left for an additional 24 hours. Cells were fixed with 100%methanol for 10 minutes and then washed three times with PBS. Coverslipswere blocked for 1 hour in 2% BSA/PBS and incubated for a further 1 hourin the presence of mouse anti-E-cadherin antibody (Transduction Labs,Lexington, Ky.; 1:250 dilution). Primary and secondary antibodies werediluted in 2% BSAIPBS. Following incubation in the primary antibody,coverslips were washed three times for 5 minutes each in PBS andincubated for 1 hour with donkey anti-mouse antibody conjugated tofluorescein (Jackson Immuno Research, West Grove, Pa.; diluted 1:200).Following a further wash in PBS (3×5 min) coverslips were mounted andviewed by confocal microscopy.

The peptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) disrupted NRK cell adhesionFIG. 11D, compare to 11A), whereas N-Ac-CHGVC-NH₂ (SEQ ID NO:11) had noaffect on cell adhesion (FIG. 11C). In the presence of N-Ac-CHGVC-NH₂(SEQ ID NO:11), the NRK cells formed tightly adherent monolayers with acobblestone morphology. They also expressed E-cadherin, as judged byimmunofluorescent staining protocols (Laird et al., J. Cell Biol.131:1193-1203, 1995) (FIG. 12C). In contrast, the NRK cells which weretreated with N-Ac-CHAVC-NH₂ (SEQ ID NO:10) did not adhere to one anotherand failed to form a contiguous monolayer (FIG. 11D). Furthermore, thesecells expressed greatly reduced levels of E-cadherin (FIG. 12D). Thesedata demonstrate that N-Ac-CHAVC-NH₂ (SEQ ID NO:10) is capable ofdisrupting NRK cell adhesion.

EXAMPLE 7 Enhancement of Human Skin Permeability

The epithelial cells of the skin (known as keratinocytes) expressE-cadherin. This Example illustrates the use of a representative cyclicpeptide to enhance the permeability of human skin.

Abdominal skin from humans at autopsy within 24 hours of death was usedin these assays. The effect of N-Ac-CHAVC-NH₂ (SEQ ID NO:10) andN-Ac-CHGVC-NH₂ (SEQ ID NO:11), used at a concentration of 500 μg/ml or2.5 mg/ml, on the penetration of two fluorescent markers, Oregon Green488 (charge −1, MW 386 daltons) and Rhodamine Green 3000 Dextran (nocharge, MW 3000 daltons) through human skin was then evaluated utilizinga Franz Cell apparatus (Franz, Curr. Prob. Dermatol. 7:58-68, 1978;Franz, J. Invest. Dermatol. 64:190-195, 1975). The peptides and markerswere dissolved in sterile phosphate buffer, pH 7.2, and phosphate bufferwas used as the receptor fluid. 150 μl of solution containing 0.2 mgOregon Green and 1.0 mg Rhodamine Green was used to evaluate 500 μg/mlpeptide; 200 μl of solution containing 0.05 mg Oregon Green and 1.250 mgRhodamine Green was used to evaluate 2.5 mg/ml peptide. The solution wasplaced on top of the epidermal side of the skin, and the penetration ofthe markers through the skin was assessed using a fluorescentspectrophotometric method (in a Perkin Elmer 650-105 FluorescenceSpectrophotometer, and comparing the reading to a standard curve) at 6,12, 24, 36, and 48 hours after the start of the experiment. Thefluorescent units were converted to a concentration unit of microgram/5ml (volume of the receiver compartment) using a standard curve andregression analysis equations. The curve was linear for theconcentrations tested for both markers (r²=1 for OrG and 0.997 for RhG).For each peptide and marker combination, the experiment was performed intriplicate.

At 500 μg/ml, N-Ac-CHAVC-NH₂ (SEQ ID NO:10; sample #1) slightlyincreased the penetration of Oregon Green through the skin, as comparedto the effect of N-Ac-CHGVC-NH₂ (SEQ ID NO:11; sample #3) on thepenetration of this marker (Table 3 and FIG. 16). The penetration ofRhodamine Green through the skin was significantly increased in thepresence of N-Ac-CHAVC-NH₂ (SEQ ID NO:10), in comparison toN-Ac-CHGVC-NH₂ (SEQ ID NO:11) (Table 4 and FIG. 17).

At 2.5 mg/ml, N-Ac-CHAVC-NH₂ (SEQ ID NO:10; sample #1) increased thepenetration of Oregon Green through the skin, as compared to the effectof N-Ac-CHGVC-NH₂ (SEQ ID NO:11; sample #3) on the penetration of thismarker (Table 3 and FIG. 18). The penetration of Rhodamine Green throughthe skin was significantly increased in the presence of N-Ac-CHAVC-NH₂(SEQ ID NO:10), in comparison to N-Ac-CHGVC-NH₂ (SEQ ID NO:11) (Table 4and FIG. 19).

TABLE 3 *Percutaneous absorption concentration (mg/5 ml) for OregonGreen ™ 488 as a function of time t = t = t = t = t = 6 12 24 36 48#Sample# hours hours hours hours hours 500 μg/ml Peptide 1Sample#1 0.0280.096 0.470 0.544 0.665 2Sample#1 0.167 0.322 1.096 1.56 1.725 3Sample#10.058 0.352 0.773 0.902 0.971 Mean Sample#1 0.084 0.225 0.780 1.00 1.1201Sample#3 0.098 0.200 0.709 0.769 0.923 2Sample#3 0.022 0.107 0.8640.923 1.021 3Sample#3 0.045 0.088 0.522 0.714 0.764 Mean Sample#3 0.0550.132 0.698 0.802 0.902 2.5 mg/ml Peptide 1Sample#1 0.14 0.44 0.67 0.760.83 2Sample#1 0.11 0.32 0.33 0.88 0.56 3Sample#1 0.16 0.45 0.63 0.991.06 Mean Sample#1 0.14 0.40 0.54 0.88 0.82 1Sample#3 0.04 0.11 0.120.23 0.36 2Sample#3 0.01 0.04 0.11 0.22 0.26 3Sample#3 0.06 0.08 0.260.29 0.46 Mean Sample#3 0.04 0.07 0.16 0.25 0.36 no dye 0 0 0 0 0 no dye0 0 0 0 0 *Defined as amount found in the receiver compartment (volume =5 ml)

TABLE 4 *Percutaneous absorption concentration (mg/5 ml) for DextranRhodamine Green 3000 as a function of time t = t = t = t = t = 6 12 2436 48 #Sample# hours hours hours hours hours 500 μg/ml Peptide 1Sample#1 0.4  3.0 16.174  21.044  25.747 2Sample#1  0.8  2.0  4.074  5.556 6.481 3Sample#1  1.2  5.556 13.158  17.565  27.826 Mean Sample#1  0.8 3.52 11.15  14.72  20.02 1Sample#3  0.2  0.6  1.0  1.0  1.8 2Sample#3 0.3  1.0  1.4  1.6  5.370 3Sample#3  0.2  0.4  0.8  1.0  1.8 MeanSample#3  0.23  0.67  1.07  1.2  2.99 2.5 mg/ml Peptide 1Sample#1 24.5245.35 66.28 120.0 146.79 2Sample#1  2.4 25.22 35.22  42.36  47.003Sample#1 11.05 23.83 44.85  51.50  60.1 Mean Sample#1 12.66 31.47 48.78 71.28 133.56 1Sample#3  1.8 17.02 27.47  33.06  40.86 2Sample#3  0.2 2.0  5.56  5.79  8.25 3Sample#3  3.8  7.89 13.9  20.35  27.48 MeanSample#3  1.93  8.97 15.64  19.73  25.53 no dye  0  0  0  0  0 no dye  0 0  0  0  0 *Defined as amount found in the receiver compartment (volume= 5 ml)

EXAMPLE 8 Disruption of Human Ovarian Cancer Cell Adhesion

This Example further illustrates the ability of representative cyclicpeptides to disrupt human ovarian cancer cell adhesion.

The human ovarian cancer cell line OVCAR-3, which expresses E-cadherin,was used in these experiments. Cells were cultured in RPMI supplementedwith insulin and containing 20% FCS. Cells were grown in T-250 cultureflasks and maintained by periodic subculturing. Cells were harvestedfrom flasks and seeded in individual wells of 96-well culture dishes(surface area of each well was 0.32 cm²) at a density of 50,000 cellsper well in 0.1 ml media containing the cyclic peptides (atconcentrations of 1, 0.1, or 0.01 mg/ml). Media control wells were alsoestablished. Cultures were evaluated periodically by microscopicexamination under both bright field and phase contrast conditions, andwere maintained for 48 hours. N-Ac-CHAVC-NH₂ (SEQ ID NO:10) was found tobe inactive within this assay at these concentrations. However, thecyclic peptide N-Ac-CHAVSC-NH₂ (SEQ ID NO:38) disrupted OVCAR-adhesion(FIGS. 13A-C)). This data demonstrates that N-Ac-CHAVSC-NH₂ (SEQ IDNO:38) specifically affects cells that express E-cadherin.

EXAMPLE 9 Disruption of Melanoma Cell Adhesion

This Example illustrates the ability of a representative cyclic peptideto disrupt melanoma cell adhesion.

Melanoma ME115 cells (kindly provided by Meenhard Herlyn, WistarInstitute, Philadelphia, Pa.) were plated on glass coverslips andcultured for 24 hours in 50% keratinocyte growth medium (Clonetics, SanDiego, Calif.) and 50% L15. Fresh medium containing the cyclic peptides(final concentration 500 μg/mL media) N-Ac-CHAVC-NH₂ (SEQ ID NO:10) orN-Ac-CHGVC-NH₂ (SEQ ID NO:11) was then added. Following 24 hours ofculture in the presence of the peptides, the medium was removed andfresh medium containing the peptides was added. The cells were fixed 24hours later with cold methanol and stored in phosphate buffered saline(PBS).

Coverslips were blocked for 1 hour in 3% ovalbumin/PBS and incubated fora further 1 hour in the presence of rabbit pan-cadherin antibody (SigmaChemical Co., St. Louis, Mo.) diluted 1:500. Primary and secondaryantibodies were diluted in PBS containing 6% normal goat serum.Following incubation in the primary antibody, coverslips were washed 3times for 5 minutes each in PBS and incubated for 1 hour in goatanti-rabbit immunoglobulin G conjugated to fluorescein (Kiekegard andPerry, South San Francisco, Calif.) diluted 1:100. Following a furtherwash in PBS (3×5 minutes) coverslips were mounted in Vectashield (VectorLabs, Burlingame, Calif.) and viewed with a Zeiss infinity correctedmicroscope.

Photographs, shown in FIG. 14, show an absence of cell membrane stainingand the appearance of bright intracellular vesicular staining in cellstreated with N-Ac-CHAVC-NH₂ (SEQ ID NO:10). In contrast, cells exposedto N-Ac-CHGVC-NH₂ (SEQ ID NO:11) displayed cadherin staining all overthe cell membrane. Occasionally, the staining concentrated at points ofcell-cell contact. These results indicate that the representative cyclicpeptide N-Ac-CHAVC-NH₂ (SEQ ID NO:10) disrupts melanoma cell adhesion.

EXAMPLE 10 Disruption of Breast Cancer Cell Adhesion

This Example illustrates the ability of a representative cyclic peptideto disrupt human breast epithelial cell adhesion.

A1N4 human breast epithelial cells (kindly provided by Martha Stampfer,Lawrence Berkeley Laboratory, Berkeley, Calif.) were plated on glasscoverslips and cultured in F12/DME containing 0.5% FCS and 10 ng/mL EGFfor 24 hours. Fresh medium containing the cyclic peptides (finalconcentration 500 μg/mL media) N-Ac-CHAVC-NH₂ (SEQ ID NO: 10) orN-Ac-CHGVC-NH₂ (SEQ ID NO:1) was then added. Following 24 hours ofculture in the presence of the peptides, the medium was removed andfresh medium containing the peptides was added. The cells were fixed 24hours later with cold methanol and stored in phosphate buffered saline(PBS).

Coverslips were blocked for 1 hour in 3% ovalbumin/PBS and incubated fora further 1 hour in the presence of 1 μg/mL mouse anti-E-cadherinantibody (Zymed, Gaithersburg, Md.). Primary and secondary antibodieswere diluted in PBS containing 6% normal goat serum. Followingincubation in the primary antibody, coverslips were washed 3 times for 5minutes each in PBS and incubated for 1 hour with goat anti-mouseconjugated to fluorescein (Kiekegard and Perry, South San Francisco,Calif.) diluted 1:100. Following a further wash in PBS (3×5 minutes)coverslips were mounted in Vectashield (Vector Labs, Burlingame, Calif.)and viewed with a Zeiss infinity corrected microscope.

Photographs, shown in FIGS. 15A and 15B, show reduced E-cadherinstaining with a stitched appearance in cells treated with N-Ac-CHAVC-NH₂(SEQ ID NO:10). In addition, holes are present in the monolayer wherethe cells have retracted from one another. In contrast, cells exposed toN-Ac-CHGVC-NH₂ (SEQ ID NO:11) displayed E-cadherin staining concentratedat points of cell-cell contact and formed a tightly adherent monolayer.

EXAMPLE 11 Toxicity and Cell Proliferation Studies

This Example illustrates the initial work to evaluate the cytotoxiceffects of representative cyclic peptides.

N-Ac-CHAVC-NH₂ (SEQ ID NO:10) and the control peptide N-Ac-CHGVC-NH₂(SEQ ID NO:11) were evaluated for possible cytotoxic effects on humanmicrovascular endothelial (HMVEC; Clonetics), human umbilical veinendothelial (HUVEC; ATCC #CRL-1730), IAFp2 (human fibroblast cell line;Institute Armand-Frapier, Montreal, Quebec), WI-38 (human fibroblastcell line; ATCC #CCL-75), MDA-MB231 (human breast cancer cell line; ATCC#HTB-26), and PC-3 (human prostate cancer cell line; ATCC #CRL-1435)cells utilizing the MTT assay (Plumb et al., Cancer Res. 49:4435-4440,1989). Neither of the peptides was cytotoxic at concentrations up to andincluding 100 μM. Similarly, neither of the peptides was capable ofinhibiting the proliferation of the above cell lines at concentrationsup to 100 μM, as judged by ³H-thymidine incorporation assays.

In fact, none of the compounds tested thus far show any cytotoxicity atconcentrations up to and including 100 μM (Table 5 and 6). However,N-Ac-CHAVSC-NH₂ (SEQ ID NO:38), N-Ac-CHGVSC-NH₂ (SEQ ID NO:39),N-Ac-CVAHC-NH₂ (SEQ ID NO:18), N-Ac-CVGHC-NH₂ (SEQ ID NO:19) andN-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42) inhibited the proliferation of HUVEC atconcentrations (IC₅₀ values) of 57 μM, 42 μM, 8 μM, 30 μM and 69 μMrespectively, as judged by ³H-thymidine incorporation assays.N-Ac-CSHAVSSC-NH₂ (SEQ ID NO:42) also inhibited the proliferation ofMDA-MB231 cells at a concentration of 76 μM and HMVEC cells at aconcentration of 70 μM (Tables 5 and 6). N-Ac-CHAVSC-NH₂ (SEQ ID NO:38)inhibited the proliferation of MDA-MB231 cells at a concentration of 52μM.

TABLE 5 Evaluation of Peptides for Cytotoxicity and Capacity to InhibitCell Proliferation of Normal Cells (IC₅0 in μM) Normal Cells HMVEC HUVECIAFp2 WI-38 SEQ Cell Cell Cell Cell Peptide ID prol Cytotox Prol CytotoxProl Cytotox Prol Cytoto N—Ac-CHGVC-NH₂ 11 >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100μ (control for #1) N—Ac-CHAVC-NH₂ 10 >100μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μ (#1)H-CHGVC-NH₂ 11 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100μ (control for #2) H-CHAVC-NH₂ (#2) 10 >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100μ N—Ac-CHGVSC-NH₂ 39 >100 μM >100μM  42 μM >100 μM >100 μM >100 μM >100 μM >100μ (control for #18)N—Ac-CHAVSC-NH₂ 38 >100 μM >100 μM  57 μM >100 μM >100 μM >100 μM >100μM >100μ *(#18) N—Ac-CSHGVC-NH₂ 37 >100 μM >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100μ (control for #16) N—Ac-CSHAVC-NH₂ 36 >100μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μ (#16)N—Ac-CAHGVDC- 27 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100μ NH₂ (control for #22) N—Ac-CAHAVDC- 26 >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100μ NH₂ (#22) N—Ac-KHGVD-NH₂13 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μ(control for #26) N—Ac-KHAVD-NH₂ 12 >100 μM >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100μ (#26) H-CAHGVDC-NH₂ 27 >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100μ (control for #45) H-CAHAVDC-NH₂26 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μ (#45)H-CAHGVDIC-NH₂ 25 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100μ (control for #47) H-CAHAVDIC-NH₂ 24 >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100 μM >100μ (#47) N—Ac-CVGHC-NH₂ 19 >100μM >100 μM  30 μM >100 μM >100 μM >100 μM >100 μM >100μ (control for#32) N—Ac-CVAHC-NH₂ 18 >100 μM >100 μM   8 μM >100 μM >100 μM >100μM >100 μM >100μ (#32) N—Ac-CAHGVDIC- 25 >100 μM >100 μM >100 μM >100μM >100 μM >100 μM >100 μM >100μ NH₂ (control for #14) N—Ac-CAHAVDIC-24 >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100 μM >100μ NH₂(#14) N—Ac-CSHGVSSC- 43 >100 μM >100 μM >100 μM >100 μM >100 μM >100μM >100 μM >100μ NH₂ (control for #24) N—Ac-CSHAVSSC- 42  70 μM >100 μM 69 μM >100 μM >100 μM >100 μM >100 μM >100μ NH₂* (#24) *Incompletelysoluble in RPMI at 1 mM

TABLE 6 Evaluation of Peptides for Cytotoxicity and Capacity to InhibitCell Proliferation of Tumoral Cells (IC₅0 in μM) Tumoral Cells SEQMDA-MB231 PC-3 Peptide ID Cell Prol Cytotox Cell Prol CytotoxN—Ac-CHGVC-NH₂ (control 11 >100 μM >100 μM >100 μM >100 μM for #1)N—Ac-CHAVC-NH₂ (#1) 10 >100 μM >100 μM >100 μM >100 μM H-CHGVC-NH₂(control for 11 >100 μM >100 μM >100 μM >100 μM #2) H-CHAVC-NH₂ (#2)10 >100 μM >100 μM >100 μM >100 μM N—Ac-CHGVSC-NH₂ 39 >100 μM >100μM >100 μM >100 μM (control for #18) N—Ac-CHAVSC-NH₂* (#18) 38  52μM >100 μM >100 μM >100 μM N—Ac-CSHGVC-NH₂ 37 >100 μM >100 μM >100μM >100 μM (control for #16) N—Ac-CSHAVC-NH₂ (#16) 36 >100 μM >100μM >100 μM >100 μM N—Ac-CAHGVDC-NH₂ 27 >100 μM >100 μM >100 μM >100 μM(control for #22) N—Ac-CAHAVDC-NH₂ 26 >100 μM >100 μM >100 μM >100 μM(#22) N—Ac-KHGVD-NH₂ 13 >100 μM >100 μM >100 μM >100 μM (control for#26) N—Ac-KHAVD-NH₂ (#26) 12 >100 μM >100 μM >100 μM >100 μMH-CAHGVDC-NH₂ 27 >100 μM >100 μM >100 μM >100 μM (control for #45)H-CAHAVDC-NH₂ (#45) 26 >100 μM >100 μM >100 μM >100 μM H-CAHGVDIC-NH₂25 >100 μM >100 μM >100 μM >100 μM (control for #47) H-CAHAVDIC-NH₂(#47) 24 >100 μM >100 μM >100 μM >100 μM N—Ac-CVGHC-NH₂ 19 >100 μM >100μM >100 μM >100 μM (control for #32) N—Ac-CVAHC-NH₂ (#32) 18 >100μM >100 μM >100 μM >100 μM N—Ac-CAHGVDIC-NH₂ 25 >100 μM >100 μM >100μM >100 μM (control for #14) N—Ac-CAHAVDIC-NH₂ 24 >100 μM >100 μM >100μM >100 μM (#14) N—Ac-CSHGVSSC-NH₂ 43 >100 μM >100 μM >100 μM >100 μM(control for #24) N—Ac-CSHAVSSC-NH₂* 42  76 μM >100 μM >100 μM >100 μM(#24) *Incompletely soluble in RPMI at 1 mM

EXAMPLE 12 Chronic Toxicity Study

This Example illustrates a toxicity study performed using arepresentative cyclic peptide.

Varying amounts of H-CHAVC-NH₂ (SEQ ID NO:10; 2 mg/kg, 20 mg/kg and 125mg/kg) were injected into mice intraperitoneally every day for threedays. During the recovery period (days 4-8), animals were observed forclinical symptoms. Body weight was measured (Table 22) and nosignificant differences occurred. In addition, no clinical symptoms wereobserved on the treatment or recovery days. Following the four dayrecovery period, autopsies were performed and no abnormalities wereobserved.

EXAMPLE 13 Stability of Cyclic Peptide in Blood

This Example illustrates the stability of a representative cyclicpeptide in mouse whole blood.

50 μl of a stock solution containing 12.5 μg/ml H-CHAVC-NH₂ (SEQ IDNO:10) was added to mouse whole blood and incubated at 37° C. Aliquotswere removed at intervals up to 240 minutes, precipitated withacetonitrile, centrifuged and analyzed by HPLC. The results (Table 7 andFIG. 21) are expressed as % remaining at the various time points, andshow generally good stability in blood.

TABLE 7 Stability of Representative Cyclic Peptide in Mouse Whole BloodTime Area Area % (Min.) 1 2 Average Remaining  0 341344 246905  294124.5 100.00 10 308924 273072 290998  98.94 20 289861 220056  254958.5  86.68 30 353019 310559 331789 112.81 45 376231 270860  323545.5 110.00 60 373695 188255 280975  95.53 90 435555 216709 326132110.88 120  231694 168880 200287  68.10 240  221952 242148 232050  78.90

EXAMPLE 14 Use of Flanking Sequences to Influence Cadherin ReceptorSpecificity

This Example illustrates the effect of sequences that flank the HAVsequence on specificity for N-cadherin-mediated responses.

Cell culture and neurite outgrowth assays. Co-cultures of cerebellarneurons on monolayers of control 3T3 cells and monolayers of transfected3T3 cells that express physiological levels of chick N-cadherin or humanL1 were established as previously described (Williams et al., Neuron13:583-594, 1994). In brief, 80,000 3T3 cells (control and transfected)were plated into individual chambers of an eight-chamber tissue cultureslide coated with polylysine and fibronectin and cultured in DMEM/10%FCS. After 24 hours, when confluent monolayers had formed, the mediumwas removed and 3000 cerebellar neurons isolated from post-natal day 2-3rats were plated into each well in SATO media (Doherty et al., Nature343:464-466, 1990) supplemented with 2% FCS. All of the test peptideswere added immediately before the neurons as a 2X stock prepared inSATO/2% FCS. The co-cultures were maintained for 16-18 hours, at whichtime they were fixed and immunostained for GAP-43 which is present onlyin the neurons and delineates the neuritic processes. The mean length ofthe longest neurite per cell was measured for 150-200 neurons sampled inreplicate cultures as previously described (Williams et al., Neuron13:583-594, 1994). The percentage inhibition of neurite outgrowth atvarious peptide concentrations was calculated as the average of at leastthree independent experiments. Dose-response curves were evaluated andthe EC₅₀ values determined.

Peptide Synthesis. All peptides were synthesized using the solid-phasemethod (Merrifield, Journal of the American Chemical Society 85:2149,1963; Stewart and Young, (1969) Solid Phase Peptide Synthesis, W. H.Freeman, San Francisco). The peptides were assembled onmethylbenzhydrylamine resin for the C-terminal amide peptides and thetraditional Merrifield resins were used for the C-terminal acidpeptides. Acetylation of the N-terminal was performed by reacting thepeptide resins with a solution of acetic anhydride in dichloromethane inthe presence of diisopropylethylamine after removal of the N-α-Boc byacidolysis using trifluoroacetic acid. All of the cyclic peptides bearthe disulfide tether Cys-S-S-Cys. Cyclization was accomplished byreacting the side chain thiol functionalities of the two cysteineresidues with a 10% solution of iodine in methanol.

All peptides with the exception of N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50) wereprepared as a stock solution at a concentration of 5-10 mg/ml indistilled water, and stored in small aliquots at −70° C. until needed.For solubility reasons N-Ac-CHAVDIC-H₂ (SEQ ID NO:50) was made up intissue culture DMSO at a concentration of 20 mg/mL.

Effects of cyclic HAV peptides on N-cadherin function. The ability ofN-Ac-CHAVC-NH₂ (SEQ ID NO:10; compound 1) to inhibit neurite outgrowthwas initially tested. This cyclic peptide has the cadherin CAR sequence(HAV) and no flanking amino acid residues. Neurons were cultured onconfluent monolayers of control (untransfected) and N-cadherinexpressing 3T3 cells for 16-18 hours. The cells were then fixed and thelength of the longest neurite on 150-200 neurons was determined bystandard assay, as described above. FIG. 22 gives the mean neuritelength in a representative experiment where cerebellar neurons have beencultured over control and N-cadherin expressing cells. In the absence ofpeptide, the mean length of the longest neurite per cell wasapproximately double on the N-cadherin expressing cells, as compared to3T3 cells. This response requires N-cadherin function in both the neuronand transfected fibroblast. FIG. 22 also illustrates inhibition ofneurite outgrowth in neurons cultured over N-cadherin expressing cellsin the presence of N-Ac-CHAVC-NH₂ (SEQ ID NO:10; compound 1,500 μg/mL).In addition, the corresponding control peptide N-Ac-CHGVC-NH₂ (SEQ IDNO:11; compound 2,500 μg/mL) had no effect on neurite outgrowth overN-cadherin expressing monolayers (FIG. 22).

FIG. 23 gives the pooled data from a number of experiments where theneurons have been cultured over control and N-cadherin expressing cellsin the presence of increasing concentrations of N-Ac-CHAVC-NH₂ (SEQ IDNO:10; compound 1). This compound has no significant effect on theN-cadherin response at concentrations up to 62 μg/ml. A significantinhibition (33.2+/−4.0%) of the response was seen at a peptideconcentration of 125 μg/ml (mean+/−s.e.m, n=3 independent experiments),with a more complete inhibition at 250 μg/ml. Results pooled from fourindependent experiments demonstrated a 68.2+/−5.1% inhibition of theN-cadherin response when the peptide was present at 250 μg/ml (see Table8). An EC₅₀ value of 0.22 mM was obtained from the dose-response curve.In contrast to the effects of the peptide on neurite outgrowth overN-cadherin expressing cells, it had no significant effect on neuriteextension over control 3T3 cells (FIG. 23). This observationdemonstrates that N-Ac-CHAVC-NH₂ (SEQ ID NO:10) is capable of acting asan antagonist and inhibiting cadherin function. Additionally,N-Ac-CHAVC-NH₂ (SEQ ID NO:10) does not inhibit integrin receptorfunction, as the latter is required for neurite extension over 3T3cells. Compound 1 alone elicits a biological response of similar potencyto the linear 10-mer N-Ac-LRAHAVDING-NH₂ (SEQ ID NO:79; % inhibition at250 mg/mL, 68.8+/−4.1). In contrast, compound 3, with a free amino groupat the N-terminal region, was inactive (Table 8).

Peptides included in Table 8 are placed into one of three groups. Thefirst group, comprising compounds 1 and 3 can be viewed as potentialgeneral or non-specific cadherin inhibitors. The second group, whichincludes compounds 23, 25, 27, 29, and 31, were designed as putativeE-cadherin specific inhibitors by incorporation of flanking amino acidsfrom the HAV region of native human E-cadherin. The remainingHAV-containing compounds were designed as putative N-cadherin inhibitorsby virtue of their HAV flanking amino acids being derived from thenative human N-cadherin sequence.

Placement of amino acids derived from the N-cadherin sequence on theN-terminus of the HAV sequence appears to either have little affect(compound 7, N-Ac-CAHAVC-NH₂; SEQ ID NO:22) or a detrimental affect(e.g., compound 17, N-Ac-CLRAHAVC-NH₂; SEQ ID NO:30) on activity. Incontrast, addition of an aspartic acid residue on the C-terminus(compound 5, N-Ac-CHAVDC-NH₂; SEQ ID NO:20) dramatically increased theinhibitory activity of the peptides (Table 1). Addition of amino acidresidues on the N-terminus of the CAR sequence in compound 5 (compound11, N-Ac-CAHAVDC-NH₂, SEQ ID NO:26; compound 17, N-Ac-CRAHAVDC-NH₂; SEQID NO:28) completely eliminated inhibitory activity. Addition of asecond amino acid on the C-terminus (Ile) to yield N-Ac-CHAVDIC-NH₂(compound 33; SEQ ID NO:50) further increased activity from that foundfor compound 5 and addition of an amino acid to the N-terminus (compound13, N-Ac-CAHAVDIC-NH₂; SEQ ID NO:24) reduced, but did not eliminate, theactivity. Again removal of the N-terminus blocking group to yieldH-CAHAVDIC-NH₂ (compound 11; SEQ ID NO:24) resulted in total loss ofactivity. Further extension of the C-terminus to yield N-Ac-CHAVDINC-NH₂(compound 34; SEQ ID NO:51) resulted in only a slight loss in activityas exemplified by the small difference in the EC₅₀ values for these twocompounds (Table 9). A further addition of a glycine residue (compound35, N-Ac-CHAVDINGC-NH₂ (SEQ ID NO:76) completely abrogates activity.Furthermore, the most active N-cadherin antagonists (N-Ac-CHAVDIC-NH₂(SEQ ID NO:50) EC₅₀=0.060 mM, N-Ac-CHAVDINC-NH₂ (SEQ ID NO:51),EC₅₀=0.070 mM and N-Ac-CHAVDC-NH₂ (SEQ ID NO:20), EC₅₀=0.093 mM) did notinterfere with the ability of neurons to extend neurites over 3T3 cellsexpressing L1 at concentrations that substantially inhibited theN-cadherin response (FIG. 24).

TABLE 8 Effects of Non-Specific N-Cadherin Specific and E-CadherinSpecific Antagonists on N-Cadherin Dependent Neurite Outgrowth TestPeptide (250 μg/mL) ID % Inhibition Control Peptide (250 μg/mL) ID %Inhibition Non-Specific  1. N-Ac-CHAVC-NH₂ 10 68.2 ±  5.1 (4)  2.N-Ac-CHGVC-NH₂ 11 4.8 ± 5.3  3. H-CHAVC-NH₂ 10 1.7 ±  1.1 (3)  4.H-CHGVC-NH₂ 11 7.8 ± 7.1 N-cadherin Specific  5. N-Ac-CHAVDC-NH₂ 20 88.4±  3.7 (3)  6. N-Ac-CHGVDC-NH₂ 21 −8.6 ± 5.8  7. N-Ac-CAHAVC-NH₂ 22 58.5±  1.0 (3)  8. N-Ac-CAHGVC-NH₂ 23 −6.4 ± 5.6  9. N-Ac-CAHAVDC-NH₂ 2613.3 ±  8.3 (3) 10. N-Ac-CAHGVDC-NH₂ 27 4.0 ± 6.9 11. H-CAHAVDC-NH₂ 261.3 ± 13.0 (3) 12. H-CAHGVDC-NH₂ 27 5.7 ± 7.8 13. N-Ac-CAHAVDIC-NH₂ 2489.4 (2) 14. N-Ac-CAHGVDIC-NH₂ 25 4.8 ± 6.5 15. H-CAHAVDIC-NH₂ 24 −3.7 ± 2.9 (3) 16. H-CAHGVDIC-NH₂ 25 7.2 ± 8.1 17. N-Ac-CLRAHAVC-NH₂ 30 9.9 ± 6.6 (3) 18. N-Ac-CLRAHGVC-NH₂ 31 −0.5 ± 7.1 19. N-Ac-CRAHAVDC-NH₂ 28−5.0 ±  4.9 (3) 20. N-Ac-CRAHGVDC-NH₂ 29 −8.0 ± 6.0 21.N-Ac-CLRAHAVDC-NH₂ 32 76.3 ±  6.6 (3) 22. N-Ac-CLRAHGVDC-NH₂ 33 −6.8 ±6.2 E-cadherin Specific 23. N-Ac-CSHAVC-NH₂ 36 11.0 ±  8.6 24.N-Ac-CSHGVC-NH₂ 37 12.5 ± 7.5 25. N-Ac-CHAVSC-NH₂ 38 −2.5 ±  7.4 26.N-Ac-CHGVSC-NH₂ 39 −6.7 ± 5.8 27. N-Ac-CSHAVSC-NH₂ 40 8.3 ±  7.3 28.N-Ac-CSHGVSC-NH₂ 41 10.8 ± 7.6 29. N-Ac-CSHAVSSC-NH₂ 42 −12.6 ±  6.4 30.N-Ac-CSHGVSSC-NH₂ 43 −5.6 ± 5.9 31. N-Ac-CHAVSSC-NH₂ 44 34.4 ± 11.3 (3)32. N-Ac-CHGVSSC-NH₂ 45 14.8 ± 6.5

Structure/Activity Relationships for the Inhibition of Neurite Outgrowthwith Cyclic HA V-Containing Peptides. In order to further assess theeffects of modifying the amino acids flanking the HAV sequence onreceptor selectivity, a series of HAV-containing peptides were evaluatedfor their ability to inhibit neurite outgrowth. These peptidescorrespond to cyclized sequences derived from the human N-cadherin(RFHLRAHAVDINGN; SEQ ID NO:80) and E-cadherin (TLFSHAVSSNGN; SEQ IDNO:8 1) sequences immediately adjacent to the surrounding the activesite (HAV).

The results shown in Table 8 identify four “N-cadherin” peptides(N-Ac-CHAVDC-NH₂ (compound 5; SEQ ID NO:20), N-Ac-CAHAVC-NH₂ (compound7; SEQ ID NO:22), N-Ac-CAHAVDIC-NH₂ (compound 13; SEQ ID NO:24) andN-Ac-CLRAHAVDC-NH₂ (compound 21; SEQ ID NO:32)) which are potentinhibitors of neurite outgrowth when used at a concentration of 250μg/mL. All of these peptides except peptide N-Ac-CHAVDC-NH₂ (SEQ IDNO:20) lost activity at concentrations of 125 mg/mL or below. A doseresponse curve (FIG. 25) for N-Ac-CHAVDC-NH₂ (SEQ ID NO:20) indicatedthat significant activity remained at 33 μg/mL (% inhibition 28.5+/−10)and an EC₅₀ value of 0.093 mM was obtained. These results indicated thatthe aspartic acid on the carboxy terminus of the HAV motif was likely akey residue for N-cadherin receptor binding. To further explore theinfluence of the C-terminus residues on activity, N-Ac-CHAVDIC-NH₂(compound 33; SEQ ID NO:50), N-Ac-CHAVDINC-NH₂ (compound 34; SEQ IDNO:5 1) and N-Ac-CHAVDINGC-NH₂ (compound 35; SEQ ID NO:76) weresynthesized. Both N-Ac-CHAVDIC-NH₂ (SEQ ID NO:50) and N-Ac-CHAVDINC-NH₂(SEQ ID NO:51) turned out to be potent inhibitors (Table 9) and doseresponse curves for these two compounds yield EC₅₀ values of 0.060 mM(FIG. 26) and 0.070 mM (FIG. 27), respectively.

TABLE 9 Effect of Additional C-terminal Residues on Neurite OutgrowthSEQ EC₅₀ Test Peptide (125 μg/mL) ID % Inhibition (mM)  5.N-Ac-CHAVDC-NH₂ 20 77.1 ± 8.4 0.093 33. N-Ac-CHAVDIC-NH₂ 50 88.3 ± 7.50.060 34. N-Ac-CHAVDINC-NH₂ 51 62.0 ± 3.4 0.070 35. N-Ac-CHAVDINGC-NH₂76  1.5 ± 2.2

Interestingly, flanking of the HAV motif with amino acids found in humanE-cadherin sequence resulted in either a complete (peptides 23, 25, 27and 29) or substantial (peptide 31) reduction in inhibitory activity(Table 8). In addition, a series of corresponding control peptides, inwhich the HAV sequence had been replaced by HGV, were also tested in thescreen. All sixteen control peptides failed to inhibit the N-cadherinresponse (see Table 8). Finally, if the N-terminal blocking group wasremoved these peptides lost activity (Table 8, compounds 3, 15).

Effects of HAV-containing peptides on the L1 response. Other celladhesion molecules, such as L1, can stimulate neurite outgrowth, andthis response shares the same downstream signaling steps as theN-cadherin response. In order to ascertain the specificity of the mostactive N-cadherin antagonists (N-Ac-CHAVDC-NH₂ (compound 5; SEQ IDNO:20), N-Ac-CHAVDIC-NH₂ (compound 33; SEQ ID NO:50) andN-Ac-CHAVDINC-NH₂ (compound 34; SEQ ID NO:51)), cerebellar neurons werecultured over either control 3T3 cell monolayers, or monolayers of 3T3cells stably transfected with cDNA encoding L1 in the presence andabsence of each peptide. As previously reported, L1 stimulated neuriteoutgrowth from cerebellar neurons. This response was not inhibited byany of the above cyclic peptides at concentrations that preventedN-cadherin-mediated neurite outgrowth (FIG. 24).

These results demonstrate that cyclic HAV peptides containing flankingamino acids found in N-cadherin are potent inhibitors of neuriteoutgrowth, whereas cyclic HAV-containing peptides containing flankingamino acids found in E-cadherin are inactive for such purposes. Inaddition, specificity for the N-cadherin receptor can be built into thepeptides by adding flanking amino acids derived from native N-cadherinto the C-terminus, while addition of one or two amino acid residues onthe N-terminus appears to be detrimental to activity (addition of athird amino acid on the N-terminus to give N-Ac-CLRAHAVDC-NH₂ (compound21; SEQ ID NO:32) resulted in partial recovery of activity).Collectively, these results show that the information needed for“non-specific” cadherin binding resides in the HAV sequence, whereas therole of the surrounding amino acids is to “constrain” the side chains ofHis and Val into a conformation required for “specific” cadherin (e.g.N-cadherin) recognition.

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, thepresent invention is not limited except as by the appended claims.

81 1 108 PRT Homo sapiens 1 Asp Trp Val Ile Pro Pro Ile Asn Leu Pro GluAsn Ser Arg Gly Pro 1 5 10 15 Phe Pro Gln Glu Leu Val Arg Ile Arg SerAsp Arg Asp Lys Asn Leu 20 25 30 Ser Leu Arg Tyr Ser Val Thr Gly Pro GlyAla Asp Gln Pro Pro Thr 35 40 45 Gly Ile Phe Ile Leu Asn Pro Ile Ser GlyGln Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg Glu Gln Ile Ala Arg PheHis Leu Arg Ala His Ala 65 70 75 80 Val Asp Ile Asn Gly Asn Gln Val GluAsn Pro Ile Asp Ile Val Ile 85 90 95 Asn Val Ile Asp Met Asn Asp Asn ArgPro Glu Phe 100 105 2 108 PRT Mus musculus 2 Asp Trp Val Ile Pro Pro IleAsn Leu Pro Glu Asn Ser Arg Gly Pro 1 5 10 15 Phe Pro Gln Glu Leu ValArg Ile Arg Ser Asp Arg Asp Lys Asn Leu 20 25 30 Ser Leu Arg Tyr Ser ValThr Gly Pro Gly Ala Asp Gln Pro Pro Thr 35 40 45 Gly Ile Phe Ile Ile AsnPro Ile Ser Gly Gln Leu Ser Val Thr Lys 50 55 60 Pro Leu Asp Arg Glu LeuIle Ala Arg Phe His Leu Arg Ala His Ala 65 70 75 80 Val Asp Ile Asn GlyAsn Gln Val Glu Asn Pro Ile Asp Ile Val Ile 85 90 95 Asn Val Ile Asp MetAsn Asp Asn Arg Pro Glu Phe 100 105 3 108 PRT Bos taurus 3 Asp Trp ValIle Pro Pro Ile Asn Leu Pro Glu Asn Ser Arg Gly Pro 1 5 10 15 Phe ProGln Glu Leu Val Arg Ile Arg Ser Asp Arg Asp Lys Asn Leu 20 25 30 Ser LeuArg Tyr Ser Val Thr Gly Pro Gly Ala Asp Gln Pro Pro Thr 35 40 45 Gly IlePhe Ile Ile Asn Pro Ile Ser Gly Gln Leu Ser Val Thr Lys 50 55 60 Pro LeuAsp Arg Glu Leu Ile Ala Arg Phe His Leu Arg Ala His Ala 65 70 75 80 ValAsp Ile Asn Gly Asn Gln Val Glu Asn Pro Ile Asp Ile Val Ile 85 90 95 AsnVal Ile Asp Met Asn Asp Asn Arg Pro Glu Phe 100 105 4 108 PRT Homosapiens 4 Asp Trp Val Val Ala Pro Ile Ser Val Pro Glu Asn Gly Lys GlyPro 1 5 10 15 Phe Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp ArgAsp Thr 20 25 30 Lys Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser ProPro Glu 35 40 45 Gly Val Phe Ala Val Glu Lys Glu Thr Gly Trp Leu Leu LeuAsn Lys 50 55 60 Pro Leu Asp Arg Glu Glu Ile Ala Lys Tyr Glu Leu Phe GlyHis Ala 65 70 75 80 Val Ser Glu Asn Gly Ala Ser Val Glu Asp Pro Met AsnIle Ser Ile 85 90 95 Ile Val Thr Asp Gln Asn Asp His Lys Pro Lys Phe 100105 5 108 PRT Mus musculus 5 Glu Trp Val Met Pro Pro Ile Phe Val Pro GluAsn Gly Lys Gly Pro 1 5 10 15 Phe Pro Gln Arg Leu Asn Gln Leu Lys SerAsn Lys Asp Arg Gly Thr 20 25 30 Lys Ile Phe Tyr Ser Ile Thr Gly Pro GlyAla Asp Ser Pro Pro Glu 35 40 45 Gly Val Phe Thr Ile Glu Lys Glu Ser GlyTrp Leu Leu Leu His Met 50 55 60 Pro Leu Asp Arg Glu Lys Ile Val Lys TyrGlu Leu Tyr Gly His Ala 65 70 75 80 Val Ser Glu Asn Gly Ala Ser Val GluGlu Pro Met Asn Ile Ser Ile 85 90 95 Ile Val Thr Asp Gln Asn Asp Asn LysPro Lys Phe 100 105 6 108 PRT Homo sapiens 6 Asp Trp Val Ile Pro Pro IleSer Cys Pro Glu Asn Glu Lys Gly Pro 1 5 10 15 Phe Pro Lys Asn Leu ValGln Ile Lys Ser Asn Lys Asp Lys Glu Gly 20 25 30 Lys Val Phe Tyr Ser IleThr Gly Gln Gly Ala Asp Thr Pro Pro Val 35 40 45 Gly Val Phe Ile Ile GluArg Glu Thr Gly Trp Leu Lys Val Thr Glu 50 55 60 Pro Leu Asp Arg Glu ArgIle Ala Thr Tyr Thr Leu Phe Ser His Ala 65 70 75 80 Val Ser Ser Asn GlyAsn Ala Val Glu Asp Pro Met Glu Ile Leu Ile 85 90 95 Thr Val Thr Asp GlnAsn Asp Asn Lys Pro Glu Phe 100 105 7 108 PRT Mus musculus 7 Asp Trp ValIle Pro Pro Ile Ser Cys Pro Glu Asn Glu Lys Gly Glu 1 5 10 15 Phe ProLys Asn Leu Val Gln Ile Lys Ser Asn Arg Asp Lys Glu Thr 20 25 30 Lys ValPhe Tyr Ser Ile Thr Gly Gln Gly Ala Asp Lys Pro Pro Val 35 40 45 Gly ValPhe Ile Ile Glu Arg Glu Thr Gly Trp Leu Lys Val Thr Gln 50 55 60 Pro LeuAsp Arg Glu Ala Ile Ala Lys Tyr Ile Leu Tyr Ser His Ala 65 70 75 80 ValSer Ser Asn Gly Glu Ala Val Glu Asp Pro Met Glu Ile Val Ile 85 90 95 ThrVal Thr Asp Gln Asn Asp Asn Arg Pro Glu Phe 100 105 8 5 PRT UnknownMOD_RES (2) Where Xaa is any amino acid 8 Asp Xaa Asn Asp Asn 1 5 9 4PRT Unknown Description of Unknown Organism Cadherin Calcium BindingMotif 9 Leu Asp Arg Glu 1 10 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic Peptide with Classical Cell AdhesionRecognition Sequence 10 Cys His Ala Val Cys 1 5 11 5 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 11Cys His Gly Val Cys 1 5 12 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with cadherin cell adhesionrecognition sequence 12 Lys His Ala Val Asp 1 5 13 5 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 13Lys His Gly Val Asp 1 5 14 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with cadherin cell adhesionrecognition sequence 14 Asp His Ala Val Lys 1 5 15 5 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 15Asp His Gly Val Lys 1 5 16 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 16 Lys His Ala Val Glu 1 5 17 5 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 17Lys His Gly Val Glu 1 5 18 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 18 Cys Val Ala His Cys 1 5 19 5 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 19Cys Val Gly His Cys 1 5 20 6 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 20 Cys His Ala Val Asp Cys 1 5 21 6 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 21Cys His Gly Val Asp Cys 1 5 22 6 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 22 Cys Ala His Ala Val Cys 1 5 23 6 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 23Cys Ala His Gly Val Cys 1 5 24 8 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 24 Cys Ala His Ala Val Asp Ile Cys 1 5 25 8 PRTArtificial Sequence Description of Artificial Sequence Cyclic controlpeptide 25 Cys Ala His Gly Val Asp Ile Cys 1 5 26 7 PRT ArtificialSequence Description of Artificial Sequence Cyclic peptide withclassical cadherin cell adhesion recognition sequence 26 Cys Ala His AlaVal Asp Cys 1 5 27 7 PRT Artificial Sequence Description of ArtificialSequence Cyclic control peptide 27 Cys Ala His Gly Val Asp Cys 1 5 28 8PRT Artificial Sequence Description of Artificial Sequence Cyclicpeptide with classical cadherin cell adhesion recognition sequence 28Cys Arg Ala His Ala Val Asp Cys 1 5 29 8 PRT Artificial SequenceDescription of Artificial Sequence Cyclic control peptide 29 Cys Arg AlaHis Gly Val Asp Cys 1 5 30 8 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 30 Cys Leu Arg Ala His Ala Val Cys 1 5 31 8 PRTArtificial Sequence Description of Artificial Sequence Cyclic controlpeptide 31 Cys Leu Arg Ala His Gly Val Cys 1 5 32 9 PRT ArtificialSequence Description of Artificial Sequence Cyclic peptide withclassical cadherin cell adhesion recognition sequence 32 Cys Leu Arg AlaHis Ala Val Asp Cys 1 5 33 9 PRT Artificial Sequence Description ofArtificial Sequence Cyclic control peptide 33 Cys Leu Arg Ala His GlyVal Asp Cys 1 5 34 6 PRT Artificial Sequence Description of ArtificialSequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 34 Ala His Ala Val Asp Ile 1 5 35 6 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 35Ala His Gly Val Asp Ile 1 5 36 6 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 36 Cys Ser His Ala Val Cys 1 5 37 6 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 37Cys Ser His Gly Val Cys 1 5 38 6 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 38 Cys His Ala Val Ser Cys 1 5 39 6 PRT ArtificialSequence Description of Artificial Sequence Cyclic control peptide 39Cys His Gly Val Ser Cys 1 5 40 7 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 40 Cys Ser His Ala Val Ser Cys 1 5 41 7 PRTArtificial Sequence Description of Artificial Sequence Cyclic controlpeptide 41 Cys Ser His Gly Val Ser Cys 1 5 42 8 PRT Artificial SequenceDescription of Artificial Sequence Cyclic peptide with classicalcadherin cell adhesion recognition sequence 42 Cys Ser His Ala Val SerSer Cys 1 5 43 8 PRT Artificial Sequence Description of ArtificialSequence Cyclic control peptide 43 Cys Ser His Gly Val Ser Ser Cys 1 544 7 PRT Artificial Sequence Description of Artificial Sequence Cyclicpeptide with classical cadherin cell adhesion recognition sequence 44Cys His Ala Val Ser Ser Cys 1 5 45 7 PRT Artificial Sequence Descriptionof Artificial Sequence Cyclic control peptide 45 Cys His Gly Val Ser SerCys 1 5 46 6 PRT Artificial Sequence Description of Artificial SequenceCyclic peptide with classical cadherin cell adhesion recognitionsequence 46 Ser His Ala Val Ser Ser 1 5 47 6 PRT Artificial SequenceDescription of Artificial Sequence Cyclic control peptide 47 Ser His GlyVal Ser Ser 1 5 48 8 PRT Artificial Sequence Description of ArtificialSequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 48 Lys Ser His Ala Val Ser Ser Asp 1 5 49 8 PRTArtificial Sequence Description of Artificial Sequence Cyclic controlpeptide 49 Lys Ser His Gly Val Ser Ser Asp 1 5 50 7 PRT ArtificialSequence Description of Artificial Sequence Cyclic peptide withclassical cadherin cell adhesion recognition sequence 50 Cys His Ala ValAsp Ile Cys 1 5 51 8 PRT Artificial Sequence Description of ArtificialSequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 51 Cys His Ala Val Asp Ile Asn Cys 1 5 52 5 PRTUnknown Description of Unknown Organism Cadherin cell adhesionrecognition sequencebound by alpha-6-beta-1 integrin 52 Tyr Ile Gly SerArg 1 5 53 10 PRT Unknown Description of Unknown Organism Cadherin celladhesion recognition sequence bound by N-CAM 53 Lys Tyr Ser Phe Asn TyrAsp Gly Ser Glu 1 5 10 54 17 PRT Unknown Description of Unknown OrganismN-CAM heparin sulfate binding site 54 Ile Trp Lys His Lys Gly Arg AspVal Ile Leu Lys Lys Asp Val Arg 1 5 10 15 Phe 55 4 PRT UnknownDescription of Unknown Organism Occluding cell adhesion recognitionsequence 55 Leu Tyr His Tyr 1 56 8 PRT Unknown Description of UnknownOrganism Claudin cell adhesion recognition sequence 56 Trp Xaa Xaa XaaXaa Xaa Xaa Gly 1 5 57 9 PRT Unknown Description of Unknown OrganismNonclassical cadherin cell adhesion recognition sequence 57 Xaa Phe XaaXaa Xaa Xaa Xaa Xaa Gly 1 5 58 4 PRT Unknown Description of UnknownOrganism Representative claudin cell adhesion recognition sequence 58Ile Tyr Ser Tyr 1 59 4 PRT Unknown Description of Unknown OrganismRepresentative claudin cell adhesion recognition sequence 59 Thr Ser SerTyr 1 60 4 PRT Unknown Description of Unknown Organism Representativeclaudin cell adhesion recognition sequence 60 Val Thr Ala Phe 1 61 4 PRTUnknown Description of Unknown Organism Representative claudin celladhesion recognition sequence 61 Val Ser Ala Phe 1 62 10 PRT ArtificialSequence Description of Artificial Sequence Synthesized Peptide 62 CysAsp Gly Tyr Pro Lys Asp Cys Lys Gly 1 5 10 63 10 PRT Artificial SequenceDescription of Artificial Sequence Synthesized Cyclic Peptide 63 Cys AspGly Tyr Pro Lys Asp Cys Lys Gly 1 5 10 64 10 PRT Artificial SequenceDescription of Artificial Sequence Synthesized peptide 64 Cys Gly AsnLeu Ser Thr Cys Met Leu Gly 1 5 10 65 10 PRT Artificial SequenceDescription of Artificial Sequence Synthesized cyclic peptide 65 Cys GlyAsn Leu Ser Thr Cys Met Leu Gly 1 5 10 66 9 PRT Artificial SequenceDescription of Artificial Sequence Synthesized peptide 66 Cys Tyr IleGln Asn Cys Pro Leu Gly 1 5 67 9 PRT Artificial Sequence Description ofArtificial Sequence Synthesized cyclic peptide 67 Cys Tyr Ile Gln AsnCys Pro Leu Gly 1 5 68 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 68 Cys His Ala Val Xaa 1 5 69 10 PRT ArtificialSequence Description of Artificial Sequence Cyclic Peptide withclassical cadherin cell adhesion recognition sequence 69 Ile Xaa Tyr SerHis Ala Val Ser Cys Glu 1 5 10 70 10 PRT Artificial Sequence Descriptionof Artificial Sequence Cyclic Peptide with classical cadherin celladhesion recognition sequence 70 Ile Xaa Tyr Ser His Ala Val Ser Ser Cys1 5 10 71 9 PRT Artificial Sequence Description of Artificial SequenceCyclic peptide with classical cadherin cell adhesion recognitionsequence 71 Xaa Tyr Ser His Ala Val Ser Ser Cys 1 5 72 9 PRT ArtificialSequence Description of Artificial Sequence Cyclic peptide withclassical cadherin cell adhesion recognition sequence 72 Xaa Tyr Ser HisAla Val Ser Ser Cys 1 5 73 5 PRT Artificial Sequence Description ofArtificial Sequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 73 His Ala Val Ser Ser 1 5 74 4 PRT ArtificialSequence Description of Artificial Sequence Synthesized cyclic peptide74 Trp Gly Gly Trp 1 75 15 PRT Homo sapiens Description of ArtificialSequence Representative immunogen containing the HAV classical cadherincell adhesion recognition sequence 75 Phe His Leu Arg Ala His Ala ValAsp Ile Asn Gly Asn Gln Val 1 5 10 15 76 9 PRT Artificial SequenceDescription of Artificial Sequence Cyclic peptide with classicalcadherin cell adhesion recognition sequence 76 Cys His Ala Val Asp IleAsn Gly Cys 1 5 77 7 PRT Artificial Sequence Description of ArtificialSequence Cyclic peptide with classical cadherin cell adhesionrecognition sequence 77 Ser His Ala Val Asp Ser Ser 1 5 78 48 PRTUnknown Description of Unknown Organism Occludin cell adhesionrecognition sequnce and flanking amino acids 78 Gly Val Asn Pro Thr AlaGln Ser Ser Gly Ser Leu Tyr Gly Ser Gln 1 5 10 15 Ile Tyr Ala Leu CysAsn Gln Phe Tyr Thr Pro Ala Ala Thr Gly Leu 20 25 30 Tyr Val Asp Gln TyrLeu Tyr His Tyr Cys Val Val Asp Pro Gln Glu 35 40 45 79 10 PRTArtificial Sequence Description of Artificial Sequence Peptide withclassica l cadherin cell adhesion recognition sequence 79 Leu Arg AlaHis Ala Val Asp Ile Asn Gly 1 5 10 80 14 PRT Homo sapiens N-cadherinwith HAV cell adhesion recognition sequence and flanking amino acids 80Arg Phe His Leu Arg Ala His Ala Val Asp Ile Asn Gly Asn 1 5 10 81 12 PRTHomo sapiens E-cadherin with HAV cell adhesion recognition sequence andflanking amino acids 81 Thr Leu Phe Ser His Ala Val Ser Ser Asn Gly Asn1 5 10

What is claimed is:
 1. A method for enhancing drug delivery to thecentral nervous system of a mammal, comprising administering to a mammala cell adhesion modulating agent that inhibits cadherin-mediated celladhesion, wherein the modulating agent comprises a cyclic peptidecomprising the sequence HAV within a cyclic peptide ring that contains5-17 amino acid residues, and thereby enhancing drug delivery to thecentral nervous system of the mammal.
 2. A method according to claim 1,wherein the modulating agent comprises a sequence selected from thegroup consisting of CHAVC (SEQ ID NO:10), CHAVDC (SEQ ID NO:20), CHAVDIC(SEQ ID NO:50), CHAVDINC (SEQ ID NO:51), CHAVDINGC (SEQ ID NO:76),CAHAVC (SEQ ID NO:22), CAHAVDC (SEQ ID NO:26), CAHAVDIC (SEQ ID NO:24),CRAHAVDC (SEQ ID NO:28), CLRAHAVC (SEQ ID NO:30), CLRAHAVDC (SEQ IDNO:32), CSHAVC (SEQ ID NO:36), CHAVSC (SEQ ID NO:38), CSHAVSC (SEQ IDNO:40), CSHAVSSC (SEQ ID NO:42), HAVSSC (SEQ ID NO:44), KHAVD (SEQ IDNO:12), DHAVK (SEQ ID NO:14), KHAVE (SEQ ID NO:16), AHAVDI (SEQ IDNO:34), SHAVDSS (SEQ ID NO:77), KSHAVSSD (SEQ ID NO:48) and derivativesof the foregoing sequences having one or more C-terminal, N-terminaland/or side chain modifications.
 3. A method according to claim 1,wherein the cyclic peptide comprises an N-terminal acetyl group.
 4. Amethod according to claim 1 wherein the modulating agent furthercomprises one or more of: (a) a cell adhesion recognition sequence boundby an adhesion molecule other than a classical cadherin, wherein thecell adhesion recognition sequence is separated from any HAV sequence(s)by a linker; and/or (b) an antibody or antigen-binding fragment thereofthat specifically binds to a cell adhesion recognition sequence bound byan adhesion molecule other than a classical cadherin.
 5. A methodaccording to claim 4, wherein the cell adhesion recognition sequencecomprises a sequence selected from the group consisting of IYSY (SEQ IDNO:58), TSSY (SEQ ID NO:59), VTAF (SEQ ID NO:60), VSAF (SEQ ID NO:61)and LYHY (SEQ ID NO:55).
 6. A method according to claim 1, wherein themodulating agent is linked to a targeting agent.
 7. A method accordingto claim 1, wherein the modulating agent is linked to a drug.
 8. Amethod according to claim 1, wherein the modulating agent is presentwithin a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier.
 9. A method according to claim 8, wherein thepharmaceutical composition further comprises a modulator of celladhesion, comprising one or more of: (a) a cell adhesion recognitionsequence bound by an adhesion molecule other than a classical cadherin;and/or (b) an antibody or antigen-binding fragment thereof thatspecifically binds to a cell adhesion recognition sequence bound by anadhesion molecule other than a classical cadherin.
 10. A methodaccording to claim 9, wherein the cell adhesion recognition sequencecomprises a sequence selected from the group consisting of IYSY (SEQ IDNO:58), TSSY (SEQ ID NO:59), VTAF (SEQ ID NO:60), VSAF (SEQ ID NO:61),and LYHY (SEQ ID NO:55).